Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae

Maytham Hussein; Raad Jasim; Hakan Gocol; Mark Baker; Varsha J Thombare; James Ziogas; Aayush Purohit; Gauri G Rao; Jian Li; Tony Velkov

doi:10.1128/msphere.00537-22

. 2023 Jan 9;8(1):e00537-22. doi: 10.1128/msphere.00537-22

Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae

Maytham Hussein ^a,^#, Raad Jasim ^e,^#, Hakan Gocol ^b, Mark Baker ^c, Varsha J Thombare ^a, James Ziogas ^b, Aayush Purohit ^d, Gauri G Rao ^d, Jian Li ^a,^✉, Tony Velkov ^a,^✉

Editor: Paul M Dunman^f

PMCID: PMC9942579 PMID: 36622250

ABSTRACT

Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack.

IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as “molecular mules” for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.

KEYWORDS: polymyxin B, OMVs, proteomics, MDR Gram-negative

INTRODUCTION

Extremely drug-resistant (XDR) K. pneumoniae is a Gram-negative “superbug” responsible for numerous nosocomial and community outbreaks (1, 2). This pathogen often displays resistance toward β-lactam antibiotics (e.g., third-generation cephalosporins and carbapenems), through the production of novel extended-spectrum β-lactamases (ESBL), carbapenemases, and the New Delhi metallo-β-lactamase 1 (NDM-1) (1, 3). The upregulation of efflux pump transport proteins such as AcrAB and OqxAB is another tactic used by K. pneumoniae to extrude antibiotics from their intracellular environment, thereby preventing the accumulation of bactericidal concentrations (4). Furthermore, resistance in K. pneumoniae has arisen through the means of modifying enzymes (e.g., aminoglycoside-modifying enzymes) (5, 6).

The two clinically available polymyxins, namely, colistin and polymyxin B, have been increasingly used as the last-line therapy against problematic XDR K. pneumoniae strains (7). Surveillance reports of antimicrobial resistance indicate that 98.2% of K. pneumoniae clinical strains remain susceptible to polymyxin B and colistin (8, 9). Notwithstanding, XDR strains resistant to polymyxins have recently emerged, which underscores the need for further investigations of polymyxin resistance mechanisms in XDR K. pneumoniae (10 to 13). The primary antimicrobial killing action of polymyxins is mediated through a direct interaction with the lipid A component of lipopolysaccharide (LPS), followed by the insertion of the polymyxin molecule into the fatty acyl layer of the outer membrane (OM) (7). The integration of the polymyxin molecules into the OM results in disruption of the selective permeability barrier and leakage of intracellular contents, which culminates in bacterial death (14). Polymyxin resistance in K. pneumoniae primarily involves modification of lipid A with 4-amino-4-deoxy-l-arabinose or phosphoethanolamine (15, 16). The addition of 4-amino-4-deoxy-l-arabinose modifications to the lipid A phosphates is under the control of the two-component regulatory systems PhoPQ-PmrD-PmrAB that are activated in response to cationic antimicrobial peptides (CAMPs), low pH, low magnesium, and high iron (17). Our group has also reported that the underacylation of lipid A increases the polymyxin susceptibility of K. pneumoniae (18, 19). Outer membrane proteins (OMPs) have also been implicated in OM remodeling that is associated with increased polymyxin susceptibility in K. pneumoniae (20).

Gram-negative bacteria can shed components of their OM via outer membrane vesicles (OMVs), which are spherical bilayer structures approximately 20 to 200 nm in diameter (21). Structurally, these vesicles are made up of OM, periplasmic lipids, and proteins (22). OMVs carry bacterial components as cargo, including cytoplasmic and inner membrane proteins, genetic materials, virulence factors, metabolites, and signaling molecules (23, 24). It has been recently found that the OMVs can mediate horizontal gene transfer events and thereby, help promote the development of antibiotic resistance in Gram-negative bacteria, including K. pneumoniae (25). Therefore, OMVs play crucial roles in bacterial virulence, inflammation, host immune stimulation, and of particular interest, in defense and resistance mechanisms against antibiotics (26). OMVs protect bacteria from the effects of antibiotics through the use of antibiotic-degrading enzymes (e.g., β-lactamase and aminoglycosides-inactivating enzymes), or by capturing membrane-active antibiotics such as polymyxins (26 to 29). This is shown by the fact that incubation of susceptible strains with OMVs from polymyxin-resistant Gram-negative bacteria conferred a protective effect for susceptible bacteria against polymyxins (29). This highlights the need to understand the compositional differences in the subproteome of OMVs from polymyxin-susceptible and extremely resistant K. pneumoniae isolates. In the present study, we primarily aimed to perform a comparative analysis of the OMV subproteome of paired polymyxin-susceptible and XDR polymyxin-resistant K. pneumoniae isolates and identify key proteins that are selectively packaged from the parent bacteria into the OMV subproteome. The garnered data provide a novel understanding of the OMV subproteome associated with extreme levels of polymyxin B resistance in the problematic Gram-negative opportunistic pathogen, K. pneumoniae.

RESULTS AND DISCUSSION

OMV subproteome characterization.

Proteomic analysis of the OMVs identified 1,115 and 1,121 differentially represented proteins in the polymyxin-susceptible K. pneumoniae ATCC 700721 and its paired XDR strain, respectively. Subcellular localization and virulence prediction of the OMV subproteomes are explained in detail in the supplemental document (Text S1; Fig. S1 and S2). Comparative analysis (≥1-log_2-fold change [FC]; P ≤ 0.05; FDR ≤ 0.05) displayed that a total of 648 proteins (271 downregulated and 377 upregulated proteins) were significantly changed in the OMVs of the susceptible strain, compared to 232 significantly changed proteins (116 downregulated and 116 upregulated proteins) in the OMVs from the resistant strain (Fig. 1). Polymyxin B treatment induced 15 and 16 uniquely perturbed OMVs proteins in the OMV subproteomes of polymyxin-susceptible K. pneumoniae ATCC 700721 and polymyxin-resistant K. pneumoniae ATCC 700721R, respectively (Table 1; Fig. S3). As elaborated below, the differentially expressed OMV proteins were involved in cell envelope biosynthesis (LPS, peptidoglycan, O-antigen), protein export systems, two-component systems (TCS), cationic antimicrobial peptide (CAMP) resistance proteins, β-lactam resistance, quorum sensing, RNA degradation, nucleotide excision/repair, and protein export.

FIG 1 — Number of significantly changed OMVs proteins from *K. pneumoniae* ATCC 700721 and *K. pneumoniae* ATCC 700721R after polymyxin B therapy (n = 3). *, >1.0-log₂FC, *P ≤* 0.05; FDR ≤ 0.05.

TABLE 1.

Unique OMVs proteins perturbed in polymyxin-susceptible K. pneumoniae ATCC 700721 and polymyxin-resistant K. pneumoniae ATCC 700721R following polymyxin B treatment

Protein ID	Gene	Protein	Subcellular localisation
K. pneumoniae ATCC 700721
A6T5B0	proC	Pyrroline-5-carboxylate reductase	Cytoplasmic
A6T7E0	KPN78578	UPF0502 protein KPN78578_10500	Cytoplasmic
A6T7H8	ycfX	N-acetyl-d-glucosamine kinase	Cytoplasmic
A6T7T9	ydjQ	Endonuclease in nucleotide excision repair	Cytoplasmic
A6T7X7	yciL	Pseudouridine synthase	Cytoplasmic
A6T7G4	lpoB	Penicillin-binding protein activator LpoB (PBP activator LpoB)	Cytoplasmic membrane
A6T4X9	bamA	Outer membrane protein assembly factor BamA	Outer membrane
A6T5E4	tsx	Nucleoside-specific channel-forming protein Tsx	Outer membrane
A6T631	fepA	Outer membrane porin, receptor for ferric enterobactin and colicins B and D	Outer membrane
A6T751	ompA	Outer membrane protein A	Outer membrane
A6T4Y0	hlpA	Periplasmic molecular chaperone for outer membrane proteins	Periplasmic
A6T509	mltD	Lytic murein transglycosylase C, membrane-bound	Periplasmic
A6T5J7	ybaY	Glycoprotein/polysaccharide metabolism	Periplasmic
A6T5R7	KPN_00488	5-hydroxyisourate hydrolase (HIU hydrolase) (HIUHase) (EC 3.5.2.17)	Periplasmic
A6T639	fepB	Ferric enterobactin (Enterochelin) binding protein periplasmic component	Periplasmic
K. pneumoniae ATCC 700721R
A6T8I9	paaJ	Acetyl-CoA acetyltransferase	Cytoplasmic
A6TCI7	rpoE	RNA polymerase sigma-70 factor	Cytoplasmic
A6TCL4	rplS	50S ribosomal protein L19	Cytoplasmic
A6TEY0	rpsL	30S ribosomal protein S12	Cytoplasmic
A6TF49	glgC	Glucose-1-phosphate adenylyltransferase	Cytoplasmic
A6TFM7	rpmG	50S ribosomal protein L33	Cytoplasmic
A6TFN2	slmA	Nucleoid occlusion factor SlmA	Cytoplasmic
A6TBW5	nuoK	NADH-quinone oxidoreductase subunit K	Cytoplasmic membrane
A6TGH3	wzz	ECA polysaccharide chain length modulation protein	Cytoplasmic membrane
A6TGI6	hemX	Uroporphyrinogen III methylase	Cytoplasmic membrane
A6THX1	mdoB	Phosphoglycerol transferase I	Cytoplasmic membrane
A6TIT5	traE	F pilus assembly protein	Cytoplasmic membrane
A6TBG4	asmA	Suppressor of ompF assembly mutants	Outer membrane
A6TAE1	lpp	Murein lipoprotein	Outer membrane
A6TA04	sodB	Superoxide dismutase	Periplasmic
A6THZ1	osmY	Hyperosmotically inducible periplasmic protein	Periplasmic

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TEXT S1

Subcellular localization and virulence prediction of the OMV subproteomes. Download Text S1, DOCX file, 0.03 MB^{(26KB, docx)}.

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Lipopolysaccharide biosynthesis and modification.

Biosynthetic proteins from the LPS biosynthesis pathway were significantly perturbed in the OMVs from the polymyxin-susceptible strain following polymyxin B treatment, whereas in the OMVs from the polymyxin-resistant strain the LPS biosynthetic proteins were marginally altered (Fig. 2; Table S1 in the supplemental material). The 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA), phosphoheptose isomerase (GmhA), and bi-functional ADP-l-glycero-d-manno-heptose synthase (HldE) were significantly upregulated in the OMVs of polymyxin-susceptible ATCC 700721 following polymyxin B treatment (log₂ FC = 4.0, 9.2, and 1.3, respectively; Fig. 2; Table S1), whereas in the polymyxin-resistant OMVs, only 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA) was significantly increased following polymyxin B treatment (Table S1). The 2-dehydro-3-deoxyphosphooctonate aldolase is responsible for linking lipid A and core oligosaccharides through synthesis of Kdo (30). In most Gram-negative bacteria, bi-functional ADP-l-glycero-d-manno-heptose synthase and phosphoheptose isomerase proteins are involved in the formation of a key component of the LPS core domain as well as enhancing the bacterial tolerance toward cell-wall-damaging agents (31, 32). It has been shown that the mutation of a gene encoding phosphoheptose isomerase in Gram-negative bacteria results in a compromised outer membrane, leading to a loss of protection normally provided by LPS and increased susceptibility to polymyxins (33).

FIG 2 — Schematic diagram depicting significantly altered OMV proteins involved in the cytoplasmic LPS biosynthetic steps, LPS periplasmic transport and assembly in the polymyxin-susceptible *K. pneumoniae* ATCC 700721 following polymyxin B treatment. OM, outer membrane; IM, inner membrane. Blue rectangles, significantly inhibited proteins; red rectangles, significantly increased proteins.

TABLE S1

Significantly impacted proteins in polymyxin-susceptible K. pneumoniae ATCC 700721 and polymyxin-resistant K. pneumoniae ATCC 700721R following polymyxin B treatment (>1.0-log₂-FC, P ≤ 0.05; FDR ≤ 0.05). Download Table S1, DOCX file, 0.04 MB^{(38.6KB, docx)}.

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Two essential enzymes of the Lpx pathway (responsible for the synthesis of the Kdo2-lipid A moiety of lipid A) (34), namely, Kdo transferase (WaaA) and tetraacyldisaccharide 4′-kinase (LpxK), were perturbed only in the polymyxin-susceptible OMVs following polymyxin treatment (log₂FC = −3.1 and −8.0, respectively; Fig. 2; Table S1). Kdo transferase catalyzes the transfer of Kdo to the lipid A precursor of LPS (35), while tetraacyldisaccharide 4′-kinase mediates the sixth step of lipid A biosynthesis by catalyzing phosphorylation of tetraacyldisaccharide-1-phosphate (DSMP) at the 4′ position yielding lipid IV_A (36). Finally, the abundance of the LPS assembly protein, LapB, an essential protein that governs the proteolytic activity of FtsH toward LpxC, was substantially decreased in the OMVs of the susceptible strain following polymyxin B treatment (log₂FC = −1.8) (Fig. 2; Table S1).

The ABC (ATP Binding Cassette) transporter MsbA that transports nascent LPS in a “trap and flip” process, was significantly decreased in the OMVs of the polymyxin-susceptible strain following polymyxin treatment (log₂FC = −2.3). Moreover, a significant decline in the abundance of two essential proteins, namely, LptC (log₂FC = −3.5), and LptE (log₂FC = −2.9), of the Lpt molecular machinery (a unique device that exports LPS across the periplasm to the cell surface) (37), was observed in the OMVs of the polymyxin-susceptible strain following polymyxin treatment (Fig. 2; Table S1).

A significant decrease in the levels of LPS-modifying enzymes was evident mainly in the OMVs of the polymyxin-susceptible strain following polymyxin B treatment. In particular, a marked decrease in the undecaprenyl phosphate-alpha-l-Ara4N transferase (ArnT) was observed in OMVs of both strains (log₂FC: resistant = −1.1; susceptible = −5.8) (Fig. 2; Table S1). This transferase enzyme catalyzes the transfer of the L-Ara4N moiety to lipid A, an important step for developing polymyxin resistance (15). The LPS-modifying enzyme lipid A 1-diphosphate synthase (LpxT) was markedly decreased (log₂FC = −3.7) in the polymyxin-susceptible OMVs after polymyxin treatment (Fig. 2; Table S1). Lipid A 1-diphosphate synthase catalyzes phosphorylation of the 1-phosphate of lipid A to form 1-diphosphate (1-PP); this modification increases the net negative charge of lipid A and thereby diminishes the efficiency of the cationic phosphoethanolamine (PEtN) modification. This finding would suggest that the bacteria jettison the enzyme from the OM via OMVs to decrease its abundance and negative impact on PEtN-mediated polymyxin resistance (38, 39). Furthermore, an inner membrane enzyme that catalyzes the oxygen-dependent formation of 2-hydroxymyristate-modified lipid A, namely, acyl-hydroxylase (LpxO), was significantly decreased (log₂FC = −2.4) in the OMVs of the polymyxin-susceptible strain following polymyxin B treatment (Fig. 2; Table S1). Notably, it has previously been reported that 2-OH myristate modifications of lipid A in pathogenic Gram-negative bacteria Salmonellae promote resistance to CAMPs (e.g., polymyxins and magainin-like peptide pGLa) (40).

The decreased abundance of LPS-modifying enzymes (i.e., ArnT, LpxT, and LpxO) following polymyxin exposure, especially in the OMVs of the polymyxin-susceptible strain, appears to be counterintuitive. However, this could be construed as a mechanism whereby the bacteria generate OMVs decorated with unmodified LPS that is a more attractive target than the bacterial outer membrane that is decorated with modified LPS, resulting in negatively charged OMVs that are more effective in shielding the bacterium and acting as decoys against polymyxin treatment (29, 41 to 43).

O-antigen nucleotide sugar biosynthesis.

The O-antigen of Klebsiella spp. comprises repeating oligosaccharide subunits linked to the core antigen of LPS, and the genetics of its biosynthesis in K. pneumoniae is governed by the WB gene clusters (44, 45). In K. pneumoniae, longer O-antigen length is linked to increased polymyxin resistance and virulence (44, 46). Proteins of O-antigen nucleotide sugar biosynthesis were significantly altered in both polymyxin-susceptible and resistant strain OMVs following treatment (Table S1). In the polymyxin-susceptible strain, six essential proteins involved in O-antigen nucleotide sugar biosynthesis underwent a significant overrepresentation in the OMV subproteome following polymyxin treatment, namely, UTP-glucose-1-phosphate uridylyltransferase (GalU), UDP-galactose 4-epimerase (GalE), UDP-glucose dehydrogenase (Ugd), dTDP-4-dehydrorhamnose reductase, glucose-1-phosphate thymidylyltransferase, and dTDP-glucose 4,6-dehydratase (>2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). Notably, the O-antigen export nucleotide binding domain was significantly downregulated in OMVs of the polymyxin-susceptible strain (log₂FC = −2.64; Table S1).

Polymyxin B treatment caused a marked increase in the levels of four O-antigen biosynthetic proteins in the polymyxin-resistant OMV subproteome, including UTP-glucose-1-phosphate uridylyltransferase, UDP-galactose 4-epimerase, UDP-glucose dehydrogenase, and dTDP-4-dehydrorhamnose reductase (>1.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). In Gram-negative bacteria, UDP-glucose dehydrogenase catalyzes the oxidation of UDP-glucose to produce UDP-glucuronic acid, which is required for biosynthesis of many bacterial surface glycostructures, including Ara4N moiety, and secreted exopolysaccharides, which enable the pathogen to develop resistance toward polymyxins and other cationic antimicrobial peptides (47, 48). UDP-glucuronic acid is also an essential substrate for biosynthesis of the O-antigen, a component of the outer membrane LPS, and capsular polysaccharides (CPSs) (49). These extracellular polysaccharides are often critical virulence factors that allow the bacteria to invade and colonize the tissue of the host as well as protect the bacteria from the host immune system (e.g., phagocytosis) (50).

Two-component systems and cationic antimicrobial peptide (CAMPs) resistance.

The PmrA/PmrB two-component systems (TCS) in Gram-negative bacteria are the major regulator of LPS modifications that confer polymyxin resistance (51). PmrD is a small basic protein that serves as the nexus between the PhoP-PhoQ and PmrA-PmrB TCSs (52). PhoP controls the activation of PmrD, which can then bind to PmrA and prolong its phosphorylation state, eventually activating the expression of the PmrA-PrmB (collectively PmrAB) system to promote LPS cationic modifications and resistance to polymyxin (39).

In the OMVs of the polymyxin-susceptible strain, seven crucial components of the OmpR protein family underwent a significant downregulation following polymyxin B treatment, namely, sensor protein PhoQ, phosphate-binding protein PstS, acid phosphatase PhoC, two-component sensor protein CpxA, multidrug resistance protein MdtA, and aerobic respiration control sensor protein (arcB) (>−2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1), whereas the OmpR family negative response regulator acrA displayed a marked upregulation in response to polymyxin treatment (log₂FC = 6.4). In the OMVs from the polymyxin-resistant strain, only three key proteins of the OmpR family underwent significant downregulation following polymyxin B treatment, namely, alkaline phosphatase PhoA (log₂FC = −3.6), phosphate-binding protein PstS (log₂FC = −3.6), and putative outer membrane protein SilC (log₂FC = −2.3) (Table S1).

It is well acknowledged that the presence of CAMPs can activate PhoQ and thereby stimulate PhoP by phosphorylation, which in turn upregulates the PmrA-PmrB system. The latter TCS is known to confer resistance to polymyxins in K. pneumoniae (53). PhoQ loss-of-function mutations are also associated with a high level of polymyxin resistance in Gram-negative bacteria (54). Importantly, the downregulation of multidrug resistance protein MdtA in the OMVs of the susceptible strain might be an indirect result of the response to polymyxin B treatment, as cyclic AMP receptor protein was significantly upregulated in the OMVs of the susceptible strain and this protein is known for its ability to suppress multidrug efflux pumps (55). The OMVs of the polymyxin-susceptible strain also displayed a marked downregulation of six critical members of the NarL family, namely, outer membrane lipoprotein RcsF, cryptic nitrate reductase 2 alpha subunit, cryptic nitrate reductase 2 beta subunit, fumarate reductase flavoprotein subunit, and succinate dehydrogenase iron-sulfur subunit (>−2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1), whereas the NarL family transcriptional regulatory protein RcsB displayed a marked upregulation (log₂FC = 5.5) following polymyxin treatment. In addition to PmrA/PmrB, we detected significant perturbations of proteins from various other TCS in the OMVs of the polymyxin-susceptible strain, which were marginally perturbed in the OMVs of the polymyxin-resistant strain (Table S1). In the OMVs of the polymyxin-susceptible strain, these TCS proteins included the Zn-binding periplasmic protein (log₂FC = −7.9), ATP-binding protein of glutamate (log₂FC = −5.9), glutamate/aspartate transport protein (log₂FC = −6.9), cyclic AMP receptor protein (log₂FC = 5.1), cytochrome oxidase bd-II (log₂FC = −12.4), protein tyrosine phosphatase (log₂FC = 3.2), and pectinesterase (log₂FC = −3.2) (Table S1). Only three non-PmrA/PmrB TCS proteins from the OMV subproteome of the polymyxin-resistant strain were differentially expressed, including ATP-binding protein glutamate (log₂FC = −2.5), cyclic AMP receptor protein (log₂FC = 1.0) and pectinesterase (log₂FC = −1.3) (Table S1). The OMV subproteome in both strains underwent significant downregulation of five essential proteins involved in CAMP resistance in response to polymyxin B treatment, namely, undecaprenyl phosphate-alpha-l-Ara4N transferase (log₂FC = −1.1, resistant; log₂FC = −5.8, susceptible), copper homeostasis protein (log₂FC = −1.8, resistant; log₂FC = −7.6, susceptible), N-acetylmuramoyl-l-alanine amidase (log₂F = −4.90, resistant; log₂FC = −6.0, susceptible), thiol:disulfide interchange protein (log₂FC = −3.4, resistant; log₂FC = −12.7, susceptible), and peptidyl-prolyl cis-trans isomerase (log₂FC = −3.4, resistant; log₂FC = −9.7, susceptible) (Table S1). Overproduction of NlpE, an outer membrane lipoprotein, induces the Cpx system (e.g., CpxA), which sequentially mediates the bacterial adhesion and repairs cell envelope proteins (56). The two-component sensor protein CpxA was significantly downregulated only in the susceptible strain OMVs (log₂FC = −2.8), but not in the OMVs of the polymyxin resistance strain. Notably, the peptidoglycan biosynthetic enzyme N-acetylmuramoyl-l-alanine amidase, and three folding chaperones, namely, thiol:disulfide interchange protein, peptidyl-prolyl cis-trans isomerase, and putative enzyme ycfS (proteins downstream of the Cpx pathway), underwent a profound decline only in the polymyxin-susceptible OMVs strain (Table S1). Intriguingly, PhoQ and acridine efflux pump, a membrane fusion protein and part of the resistance-nodulation-division family of proteins (57), was markedly downregulated in the OMVs of the polymyxin-susceptible strain (log₂FC = −1.4 and −2.2, respectively) (Table S1). Moreover, other peptide transport proteins commonly involved in resistance to CAMPs and bacterial virulence, namely, peptide transport proteins sapA (log₂FC = −2.5) and sapF (log₂FC = −2.7) (58), underwent a significant decline in their abundance, but only in the OMVs of the polymyxin-susceptible strain (Table S1). It has been shown that an increase in the expression of ABC transporters encoded by the sapABCDFZ operon enhances the development of CAMP bacterial resistance by transferring the CAMPs into the cytoplasm for intracellular proteolytic degradation (58).

β-lactam resistance.

Polymyxin B treatment produced a significant downregulation in the levels of key proteins involved in β-lactam resistance of the OMV subproteomes of both strains, particularly the polymyxin-susceptible strain (>−1.0-log₂-FC, P ≤ 0.05; FDR ≤ 0.05), namely, TEM-1 β-lactamase Class A, RND multidrug efflux systems, namely, acridine efflux pump, bifunctional penicillin-binding protein 1a, and periplasmic oligopeptide-binding protein (Table S1). Notably, the OMVs of the resistant strain displayed a significant downregulation in the abundance of class A extended-spectrum β-lactamase SHV-12 (log₂FC = −2.5) and TEM-1 β-lactamase Class A (log₂FC = −2.5) in response to polymyxin B treatment (Table S1). Similarly, the levels of periplasmic oligopeptide-binding protein in the OMVs of the resistant strain were downregulated in response to polymyxin B treatment (Table S1).

Peptidoglycan biosynthesis.

Fundamental proteins involved in peptidoglycan biosynthesis underwent significant alterations, particularly in the OMVs of the polymyxin-susceptible strain (Fig. 3; Table S1). UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA), UDP-N-acetylmuramate-l-alanine ligase (MurC), and d-alanine-d-alanine ligase (Ddl) were among the essential enzymes of peptidoglycan biosynthesis that were significantly upregulated (log₂FC = 2.0, 1.0, and 2.9, respectively; Fig. 3; Table S1), whereas, undecaprenyl-PP-MurNAc-pentapeptide-UDPGlcNAc GlcNAc transferase (MurG), bifunctional penicillin-binding protein 1a (PBP1a), penicillin-binding protein 1b (PBP1b), and d-ala-d-ala carboxypeptidase (PBP 5/DD-CPase) were all significantly downregulated (log₂FC = −3.1, −2.4, −3.2, and −2.3, respectively; Fig. 3; Table S1). Furthermore, in the OMVs of the polymyxin-susceptible strain, a set of membrane-bound PBPs that mediate the final stages of peptidoglycan synthesis were significantly downregulated (59), namely, penicillin-binding protein activator LpoB (PBP activator LpoB), carboxy-terminal protease for penicillin-binding protein 3 (PBP 3), endolytic murein transglycosylase protein (YceG), penicillin-insensitive murein endopeptidase (MepA), and d-alanyl-d-alanine carboxypeptidase penicillin-binding protein 6 (DacC) (log₂FC = −9.3, −5.7, −5.7, −7.4, and −5.3, respectively; Table S1). Notably, endolytic peptidoglycan transglycosylase (RlpA), which functions to repair aberrantly formed peptidoglycan (60), was significantly increased (log₂FC = 3.7). In the OMVs of the resistant strain, polymyxin treatment induced marked perturbations in d-ala-d-ala carboxypeptidase (PBP5), carboxy-terminal protease for penicillin-binding protein 3, and endolytic peptidoglycan transglycosylase (log₂FC = 1.0, −1.9, and −3.8, respectively; Table S1). Intriguingly, the Tol-Pal system and other related proteins that mediate peptidoglycan synthesis and outer membrane constriction during cell division (61) were significantly downregulated, particularly in the susceptible strain OMV subproteome (Table S1). These included TolB (log₂FC = −2.9, resistant; log₂FC = −2.3, susceptible), TolQ (log₂FC = −2.2, susceptible), TolR (log₂FC = −1.8, susceptible), and the cell division coordinator CpoB (log₂FC = −3.7, resistant; log₂FC = −5.4, susceptible). The levels of TolA were elevated in the resistant strain OMVs (log₂FC = 4.4), whereas its abundance was markedly decreased in the OMVs of the susceptible strain (log₂FC = −11.7; Fig. 3; Table S1).

FIG 3 — Schematic diagram shows the essential proteins involved in the peptidoglycan biosynthesis and Tol-Pal system in the OMVs of polymyxin-susceptible *K. pneumoniae* ATCC 700721 significantly altered following polymyxin B treatment. Blue rectangles, significantly inhibited proteins; red rectangles, significantly increased proteins.

Overall, there appears to be a significant reduction in the peptidoglycan biosynthetic machinery within the OMVs of the susceptible strain, whereas they were mildly perturbed in the OMVs of the polymyxin resistant strain.

Protein export and outer membrane proteins (OMPs) complex biosynthesis.

The Gram-negative protein export system is composed of sophisticated protein machinery that efficiently exports intracellular proteins, such as the twin-arginine translocation (Tat) system (transports folded proteins), Sec pathway (general secretory pathways for unfolded preproteins), and YidC (folding and assembly of membrane proteins) (62). The Sec machinery comprises a complex of interrelated cytoplasmic membrane pathways, namely, SecA, SecB, SecYEG, SecDF, YajC, YidC, and SRP pathways (63). Secretion of various protein components is often paramount for bacterial pathogenicity and virulence mechanisms like biofilm formation (64). In response to the polymyxin B treatment, the Sec-dependent pathway and Tat system proteins were minimally perturbed in OMVs subproteome of the resistant strain, while they underwent a greater alteration in their abundance in the OMVs proteins of the susceptible strain (Table S1).

In the susceptible OMVs subproteome, there was a significant increase in the levels of SecB (log₂FC = 6.0) and signal recognition particle protein SRP (log₂FC = 1.5) following polymyxin B treatment (Fig. 4). In contrast, a substantial decline in the abundance of several key components of the Sec pathway was observed following polymyxin B treatment, namely, SecA, SecD, SecF, YajC, and YidC (>−2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Fig. 4; Table S1). Furthermore, an essential integrated subunit of the twin-arginine translocation system TatA protein and Signal peptidase I (Spase I), a vital enzyme required for the bacterial survival and essential for the translocation of preproteins from the cytoplasmic to periplasmic location (65), were significantly downregulated in response to polymyxin B treatment (log₂FC = −7.4 and −5.7, respectively) (Fig. 4; Table S1).

FIG 4 — Heatmap displays the main proteins involved in protein export and outer membrane proteins (OMPs) complex biosynthesis in the OMVs of polymyxin-susceptible *K. pneumoniae* ATCC 700721 and its polymyxin-resistant derivative ATCC 700721R significantly altered following polymyxin B treatment.

In the OMVs of the polymyxin-resistant strain, an upregulation of signal recognition particle protein SRP (log₂FC = 1.3) and downregulation of TatA protein were observed in response to polymyxin B treatment (log₂FC = −1.5; Table S1). Moreover, polymyxin B treatment caused a marked reduction in the levels of four fundamental proteins of a highly conserved multiprotein Bam complex in the OMVs of the polymyxin-susceptible strain, including OMP assembly factor BamA, BamC, BamD, and BamE (>−2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Fig. 4; Table S1). These proteins accelerate the folding and membrane integration of β-barrel transmembrane proteins and are involved in a range of functions, including the creation of pores to allow the passage of diverse molecules across the bacterial membrane. Their assembly is thus essential for maintaining the integrity of the cell envelope (66). Notably, the OMVs proteins of both strains had a considerable downregulation of the chaperone protein SurA, a critical enzyme required for protecting OMPs from aggregation in the periplasm (log₂FC = −7.6 and −1.7, respectively) (Fig. 4; Table S1). It is noteworthy that SurA depletion results in a marked decrease in the outer membrane density (67). The OMVs of the polymyxin-susceptible strain treated with polymyxin B displayed a significant reduction in the abundance of three essential outer membrane proteins, namely, outer membrane protein A, outer membrane protein X, and suppressor of ompF assembly mutants (>−1.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Fig. 4; Table S1). The OmpA proteins in K. pneumoniae are major membrane proteins that are crucial for the bacterial pathogenicity (adhesion and invasion), bacterial envelope integrity (via anchoring the outer membrane to the peptidoglycan), and transfer of molecules. Notably, it has been reported that the knockout of ompA in K. pneumoniae resulted in a highly polymyxin susceptible phenotype (20). Perturbations in these proteins were not detected in the OMVs of the polymyxin-resistant strain.

RNA degradation and nucleotide excision/repair.

The RNA degradosome is a multiprotein complex protein machine that mediates the degradation of mRNA (68). The OMVs of both strains exhibited marked perturbations in the levels of principle components of the RNA degradosome in response to polymyxin treatment (Table S1). In the OMVs of the polymyxin-susceptible strain, the abundance of RNase E (RNase E), ATP-dependent RNA helicase RhlB, ATP-dependent RNA helicase DeaD, polyphosphate kinase (PKK), and poly(A) polymerase I (PAP I) all significantly decreased following polymyxin B treatment (>−1.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). In contrast, three key RNA degradosome constituents were significantly increased in response to polymyxin B treatment, namely, enolase (log₂FC = 7.9), ATP-dependent 6-phosphofructokinase (log₂FC = 4.7), and the 60-kDa GroEL chaperonin (log₂FC = 6.0) (Table S1). On the other hand, in the OMVs of the polymyxin-resistant strain, polymyxin B treatment induced a considerable increase in the abundance of five principal components of the RNA degradosome, namely, polyphosphate kinase, 60-kDa GroEL chaperonin, enolase, RNase E, and ATP-dependent RNA helicase RhlB (>1.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). RNA-binding protein Hfq (an RNA degradosome regulator that binds small regulatory RNAs and mRNAs to mediate mRNA translation in response to the bacterial envelope stressors such as polymyxins) was significantly overexpressed (log₂FC = 5.7) (Table S1) (68). Notably, there was also a significant upregulation in critical proteins involved in the nucleotide excision and repair, namely, UvrABC repair system (log₂FC = 1.1) and DNA helicase (log₂FC = 4.7) (69). In the OMVs of polymyxin-susceptible strain, the UvrABC repair system was significantly downregulated following polymyxin B treatment (log₂FC = −2.9) (Table S1). Taken together, these findings would suggest that compared to its susceptible pair, resistant strain may have an enhanced ability to withstand DNA lesions.

Quorum sensing (QS).

Gram-negative bacteria employ QS by secretion of N-acyl homoserine lactones (AHLs), sometimes in conjunction with TCS, to coordinate pathogenic behaviors such as virulence factor expressions, biofilm formation, and motility (70). Although the OMV subproteomes in both strains displayed significant perturbations in the quorum-sensing proteins in response to polymyxin B treatment, the extent of the perturbations observed in the polymyxin-susceptible strain were significantly greater. In the OMV subproteome of the polymyxin-susceptible strain, the abundance of autoinducer 2-binding protein LsrB, three oligopeptide transport proteins (periplasmic oligopeptide-binding protein, oligopeptide transporter ATP-binding component, and oligopeptide transport protein), membrane protein insertase YidC, periplasmic murein tripeptide (l-Ala-gamma-d-Glut-m-DAP) permease, protein translocase subunit SecA, preprotein translocase subunit YajC, putative ABC transporter periplasmic binding protein, and high-affinity branched-chain amino acid transport protein, all were significantly downregulated in response to polymyxin treatment (>−2.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). Similarly, but to a lesser extent, five essential proteins of a quorum-sensing system underwent a considerable downregulation in the OMVs of the polymyxin resistant strain after polymyxin B treatment, namely, periplasmic oligopeptide-binding protein, permease, oligopeptide transport protein, high-affinity branched-chain amino acid transport protein, and putative ABC transporter periplasmic binding protein (>−1.0-log₂FC, P ≤ 0.05; FDR ≤ 0.05) (Table S1). Perturbations were also evident in systems involved in biofilm formation. Notably, there was a significant decrease in the levels of autoinducer 2-binding protein LsrB component of the LuxS-dependent autoinducer AI-2 system in the polymyxin-susceptible OMVs, whereas a marked elevation in the abundance of 3-hydroxy-5-phosphonooxypentane-2,4-dione thiolase was observed following polymyxin B treatment (log₂FC = 8.4) (Table S1). Furthermore, the protein-export machinery SecB protein levels were markedly upregulated in the polymyxin-susceptible OMVs after polymyxin B treatment (log₂FC = 8.4) (Table S1). Cyclic AMP receptor protein, which is involved in biofilm formation and flagellar biosynthesis for motility (71), was significantly upregulated in both strains (log₂FC = 1.0, resistant; log₂FC = 5.1, susceptible).

Conclusions.

Collectively, our findings indicate a potential role for OMVs in neutralizing the antimicrobial action of polymyxins, through the delivery of protein cargo that facilitates resistance and cellular repair processes. In response to polymyxin B exposure, both the susceptible and resistant K. pneumoniae strains responded by modifying their OMV subproteome to include proteins involved in outer membrane remodeling (LPS, O-antigen and peptidoglycan biosynthesis), CAMP resistance, β-lactam resistance, RNA degradation, and nucleotide excision/repair and quorum sensing. Overall, this study highlights the importance of OMVs as “molecular mules” for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxin B treatment.

MATERIALS AND METHODS

Reagents.

All chemicals were purchased from Sigma-Aldrich at the highest research grade, with the exception of the ultrapure water (Fluka, Castle Hill, NSW, Australia), Tris (ICN biochemicals, Castle Hill, NSW, Australia), the precast-SDS gels (NuSep Ltd., Lane Cove, NSW, Australia), and polymyxin B (Beta pharma, Shanghai, China). Stock solutions of polymyxin B (10 mg/L) were prepared in Milli-QTM water (Millipore, North Ryde, NSW, Australia) and filtered through 0.22-μm syringe filters (Sartorius, Melbourne, Vic, Australia).

Bacterial isolates and growth conditions.

K. pneumoniae ATCC 700721 (polymyxin B MIC = 0.5 mg/L) and its paired polymyxin-resistant strain K. pneumoniae ATCC 700721R (polymyxin B MIC ≥128 mg/L) were employed in this study. A polymyxin-resistant variant of K. pneumoniae ATCC 13883 (designated 13883R) was generated as previously described (72). Bacteria were stored at −80°C in tryptone soya broth (TSB, Oxoid Australia, West Heidelberg, Victoria, Australia). One day prior to each experiment, bacteria were streaked onto nutrient agar plates (Medium Preparation Unit, University of Melbourne, Victoria, Australia). Overnight cultures were prepared by inoculating one colony in 5 mL of cation-adjusted Mueller-Hinton broth (CAMHB, Oxoid), from which a 1-in-100 dilution was made in fresh broth to prepare midlogarithmic cultures with the optimal OD600 nm ~0.5. All broth cultures were incubated at 37°C in a rotary shaker (180 rpm).

Measurements of MICs.

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints for polymyxin B for Enterobacteriaceae (e.g., K. pneumoniae) are as a susceptible breakpoint of ≤2 mg/L and a resistant breakpoint >2 mg/L (73). MIC determinations for all isolates according to CLSI guidelines using broth microdilution method were performed using three replicates (74). The microtiter plates were inoculated with 100 μL of bacterial suspension (10⁶ CFU per mL) and 100 μL of serial dilutions of the polymyxin B. Cell viability was determined by sampling wells at polymyxin concentrations greater than the MIC. These samples were serially diluted in 0.9% saline solution and plated onto nutrient agar. After incubation at 37°C for 20 h, viable colonies were counted on these plates using a plate counter (Synbiosis ProtoCOL 3, Thermo-Fischer Scientific, Melbourne, Australia).

Isolation of outer membrane vesicles (OMVs).

Midlogarithmic cultures (4 L) of each isolate were grown, polymyxin B was added to the culture volume at a final concentration of 2 mg/L, and cell-free supernatants were collected through centrifugation (15 min at 10,000 × g, 4°C). The OMV-containing supernatants were filtered through 0.22-μm membrane to remove any remaining cell debris, then concentrated through a tangential filtration concentrator unit (Pall Life Science, Ann Arbor, MI) and collected using 100 kDa Pellicon filtration cassettes (Millipore, Melbourne, Australia). A portion of the supernatant was plated for growth on agar plates overnight at 37°C to make sure that the supernatant was free of bacterial cells. OMVs in the cell-free supernatants were then pelleted down by ultracentrifugation at 150,000 × g for 2 h at 4°C in a Beckman Ultracentrifuge (SW28 rotor). Purified OMVs were concentrated and resuspended in 1 mL of sterile phosphate-buffered saline (PBS). Samples were analyzed by 1D-SDS-PAGE and the protein concentrations were determined using the Bio-Rad protein assay according to the manufacturer’s protocol (Bio-Rad, Melbourne, Australia).

Ultra-high-performance liquid chromatography coupled with mass spectrometry.

For all experiments, an Ultimate3000 ultra-high-pressure liquid chromatography (UHPLC) system (Dionex, Castle Hill, Sydney) was used equipped with a ternary low-pressure mixing gradient pump (LPG-3600), a membrane degasser unit (SRD-3600), a temperature-controlled pulled-loop auto-sampler (WPS-3000T), and a temperature-controlled column oven with flow manager (FLM-3100). The UHPLC experiments were performed using the “preconcentration” setup under the following conditions: nano-column C₁₈ PepMap100, 75 μm ID × 150 mm, 3 μm, 100 Ǻ; mobile phase A: 99.9% water + 0.1% FA (vol/vol, formic acid); mobile phase B: 20/80 water/acetonitrile (ACN) (vol/vol) + 0.08% formic acid; flow rate nano-column, 400 nL/min; gradient, 2 to 40% B over 45 min, 90% B for 5 min, 4% B for 30 min; loop size, 5 μL; injection volume, 4 μL (FullLoop) by User Defined Program. The oven temperature was set to 35°C. Collision-induced dissociation (CID) experiments for peptide identification were performed using an AmaZon ETD Ion Trap (Bruker Daltonik GmbH, Australia) equipped with an online nano-sprayer spraying from a 0.090 mm inside diameter (i.d.) and 0.02 mm i.d. fused silica capillary. Fine tuning was achieved using the smart parameter setting option (SPS) for 900 m/z, compound stability 60%, and trap drive level at 100% in normal mode resulted in the following mass spectrometric parameters: dry gas temperature, 180°C; dry gas, 4.0 L min⁻¹; nebulizer gas, 0.4 bar; electrospray voltage, 4,500 V; high-voltage end-plate offset, −200 V; capillary exit, 140 V; trap drive, 57.4; funnel 1 in 100 V, out 35 V and funnel 2 in 12 V, out 3.3 V; immunofluorescent cell count (ICC) target, 500,000; maximum accumulation time, 50 ms. The sample was measured with the enhanced scan mode at 8,100 m/z per second (which allows monoisotopic resolution up to four charge stages), polarity positive, scan range from 100 to 3,000 m/z, 5 spectra averaged, and rolling average of 2. Acquired tandem mass spectra were processed in Data Analysis 4.0; deconvoluted spectra were further analyzed with BioTools 3.2 software and submitted to Mascot database search (Mascot 2.2.04, Swissprot database). The species subset was set at K. pneumoniae, parent peptide mass tolerance ±0.4 Da, fragment mass tolerance ±0.4 Da; enzyme specificity trypsin with 2 missed cleavages considered. The following variable modifications have been used: Deamidation (NQ), Oxidation (M), and carbamidomethylation (C).

Bioinformatics analysis.

The derived MS data sets on the 3D-trap system were combined into protein compilations using the Protein Extractor functionality of Proteinscape 2.1.0 573 (Bruker Daltonics, Bremen, Germany), which conserved the individual peptides and their scores, while combining them to identify proteins with much higher significance than what is achievable using individual searches. To exclude false-positive identifications, peptides with Mascot scores below 40 (selected based on manual evaluation of the MS/MS data of peptides with scores below this number) were rejected. The identified protein sequences were manually validated in BioTools (Bruker Daltonics, Bremen, Germany) on a residue-by-residue basis using the raw data to ensure accuracy. Since this strain (ATCC 700721/MGH78578) has a complete genomic map in UniProt (75), the protein name and KEGG ID for the identified proteins were determined by performing a search in UniProt and cross-referencing using MGH78578 KEGG identifiers (76). The subcellular localization of each protein was predicted using PSORTb 3.0 and Cello V2.5 (77, 78). Furthermore, VirulentPred (http://bioinfo.icgeb.res.in/virulent/) was also employed to screen for bacterial virulence protein sequences of OMV subproteome. Comparative analysis of significantly changed proteins (fold change >2 and P value <0.05; FDR ≤0.05) was performed using MaxQuant (version 1.2.2.5) (79). The protein-associated metabolic pathways of deferentially over- and underrepresented proteins were highlighted in KEGG mapper.

FIG S1

The prediction of subcellular localization of different K. pneumoniae proteins identified using PSORTb 3.0 and Cello V2.5 following polymyxin B treatment. (A) Subcellular localization of identified OMV proteins from ATCC 700721. (B) Subcellular localization of identified OMV proteins from ATCC 700721R. Download FIG S1, TIF file, 2.1 MB^{(2.1MB, tif)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

FIG S2

Subcellular localization of quantified proteins based on their enrichment or depletion in OMVs following polymyxin B treatment. Significantly up- (red) and downregulated (blue) OMV proteins in polymyxin-susceptible K. pneumoniae ATCC 700721 (A) and polymyxin-resistant K. pneumoniae ATCC 700721R (B). The proteins were sorted according to predicted subcellular localization. C, cytoplasmic; IM, inner membrane; P, periplasmic; OM, outer membrane; E, extracellular. (≥ 1.0 log₂FC, P ≤ 0.05; FDR ≤ 0.05). Download FIG S2, TIF file, 2.0 MB^{(2MB, tif)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

FIG S3

(A) OMV proteins predicted to be associated with the virulence in polymyxin-susceptible K. pneumoniae ATCC700721 and its paired polymyxin-resistant strain following polymyxin B treatment. (B) Venn diagram for the OMVs proteins presumed to be correlated with the virulence in polymyxin-susceptible K. pneumoniae ATCC 700721 and polymyxin-resistant strain (>1.0-log₂-FC, P ≤ 0.05; FDR ≤ 0.05). Download FIG S3, TIF file, 2.6 MB^{(2.6MB, tif)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

ACKNOWLEDGMENTS

G.G.R., T.V., and J.L. are supported by the National Institute of Allergy and Infectious Diseases, award number R01AI146241. J.L. is an Australian National Health and Medical Research Council (NHMRC) Principal Research Fellow, and T.V. is an Australian NHMRC Industry Career Development Level 2 Research Fellow. The content is solely the authors' responsibility and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

Contributor Information

Jian Li, Email: jian.li@monash.edu.

Tony Velkov, Email: tony.velkov@monash.edu.

Paul M. Dunman, University of Rochester

REFERENCES

1.Walsh TR, Weeks J, Livermore DM, Toleman MA. 2011. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infectious Diseases 11:355–362. doi: 10.1016/S1473-3099(11)70059-7. [DOI] [PubMed] [Google Scholar]
2.Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N. 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infectious Diseases 10:597–602. doi: 10.1016/S1473-3099(10)70143-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. 2012. Ready for a world without antibiotics? The Pensières antibiotic resistance call to action. Antimicrob Resist Infect Control 14:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Bialek-Davenet S, Criscuolo A, Ailloud F, Passet V, Jones L, Delannoy-Vieillard A-S, Garin B, Le Hello S, Arlet G, Nicolas-Chanoine M-H, Decré D, Brisse S. 2014. Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups. Emerg Infect Dis 20:1812–1820. doi: 10.3201/eid2011.140206. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Peerayeh SN, Rostami E, Siadat SD, Derakhshan S. 2014. High rate of aminoglycoside resistance in CTX-M-15 producing Klebsiella pneumoniae isolates in Tehran, Iran. Lab Med 45:231–237. doi: 10.1309/LMDQQW246NYAHHAD. [DOI] [PubMed] [Google Scholar]
6.Endimiani A, Carias LL, Hujer AM, Bethel CR, Hujer KM, Perez F, Hutton RA, Fox WR, Hall GS, Jacobs MR, Paterson DL, Rice LB, Jenkins SG, Tenover FC, Bonomo RA. 2008. Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrob Agents Chemother 52:2680–2682. doi: 10.1128/AAC.00158-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Velkov T, Roberts KD, Nation RL, Thompson PE, Li J. 2013. Pharmacology of polymyxins: new insights into an “old” class of antibiotics. Future Microbiol 8:711–724. doi: 10.2217/fmb.13.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Gales AC, Jones R, Sader HS. 2006. Global assessment of the antimicrobial activity of polymyxin B against 54 731 clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme (2001–2004). Clin Microbiol Infect 12:315–321. doi: 10.1111/j.1469-0691.2005.01351.x. [DOI] [PubMed] [Google Scholar]
9.Weterings V, Zhou K, Rossen J, van Stenis D, Thewessen E, Kluytmans J, Veenemans J. 2015. An outbreak of colistin-resistant Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae in the Netherlands (July to December 2013), with inter-institutional spread. Eur J Clin Microbiol Infect Dis 34:1647–1655. doi: 10.1007/s10096-015-2401-2. [DOI] [PubMed] [Google Scholar]
10.Bratu S, Tolaney P, Karumudi U, Quale J, Mooty M, Nichani S, Landman D. 2005. Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents. J Antimicrob Chemother 56:128–132. doi: 10.1093/jac/dki175. [DOI] [PubMed] [Google Scholar]
11.Elemam A, Rahimian J, Mandell W. 2009. Infection with panresistant Klebsiella pneumoniae: a report of 2 cases and a brief review of the literature. Clin Infect Dis 49:271–274. doi: 10.1086/600042. [DOI] [PubMed] [Google Scholar]
12.Hussein M, Wong LJM, Zhao J, Rees VE, Allobawi R, Sharma R, Rao GG, Baker M, Li J, Velkov T. 2022. Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant Klebsiella pneumoniae. Comput Struct Biotechnol J 20:1077–1087. doi: 10.1016/j.csbj.2022.02.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Hussein M, Hu X, Paulin OK, Crawford S, Zhou QT, Baker M, Schneider-Futschik EK, Zhu Y, Li J, Velkov T. 2020. Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens. Comput Struct Biotechnol J 18:2247–2258. doi: 10.1016/j.csbj.2020.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Hsu Chen CC, Feingold DS. 1973. Mechanism of polymyxin B action and selectivity toward biologic membranes. Biochemistry 12:2105–2111. doi: 10.1021/bi00735a014. [DOI] [PubMed] [Google Scholar]
15.Falagas ME, Rafailidis PI, Matthaiou DK. 2010. Resistance to polymyxins: mechanisms, frequency and treatment options. Drug Resist Updat 13:132–138. doi: 10.1016/j.drup.2010.05.002. [DOI] [PubMed] [Google Scholar]
16.Raetz CR, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 76:295–329. doi: 10.1146/annurev.biochem.76.010307.145803. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Cheng H-Y, Chen Y-F, Peng H-L. 2010. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci 17:60. doi: 10.1186/1423-0127-17-60. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Clements A, Tull D, Jenney AW, Farn JL, Kim S-H, Bishop RE, McPhee JB, Hancock REW, Hartland EL, Pearse MJ, Wijburg OLC, Jackson DC, McConville MJ, Strugnell RA. 2007. Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides. J Biol Chem 282:15569–15577. doi: 10.1074/jbc.M701454200. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Velkov T, Soon RL, Chong PL, Huang JX, Cooper MA, Azad MAK, Baker MA, Thompson PE, Roberts K, Nation RL, Clements A, Strugnell RA, Li J. 2013. Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A. Innate Immun 19:265–277. doi: 10.1177/1753425912459092. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Llobet E, March C, Giménez P, Bengoechea JA. 2009. Klebsiella pneumoniae OmpA confers resistance to antimicrobial peptides. Antimicrob Agents Chemother 53:298–302. doi: 10.1128/AAC.00657-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Schwechheimer C, Kuehn MJ. 2015. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13:605–619. doi: 10.1038/nrmicro3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Horstman AL, Kuehn MJ. 2000. Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem 275:12489–12496. doi: 10.1074/jbc.275.17.12489. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Dorward DW, Garon CF, Judd RC. 1989. Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol 171:2499–2505. doi: 10.1128/jb.171.5.2499-2505.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Lee E-Y, Bang JY, Park GW, Choi D-S, Kang JS, Kim H-J, Park K-S, Lee J-O, Kim Y-K, Kwon K-H, Kim K-P, Gho YS. 2007. Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli. Proteomics 7:3143–3153. doi: 10.1002/pmic.200700196. [DOI] [PubMed] [Google Scholar]
25.Dell’Annunziata F, Dell’Aversana C, Doti N, Donadio G, Dal Piaz F, Izzo V, De Filippis A, Galdiero M, Altucci L, Boccia G, Galdiero M, Folliero V, Franci G. 2021. Outer membrane vesicles derived from Klebsiella pneumoniae are a driving force for horizontal gene transfer. Int J Mol Sci 22:8732. doi: 10.3390/ijms22168732. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Manning AJ, Kuehn MJ. 2011. Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol 11:258. doi: 10.1186/1471-2180-11-258. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Ciofu O, Beveridge TJ, Kadurugamuwa J, Walther-Rasmussen J, Høiby N. 2000. Chromosomal β-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. J Antimicrob Chemother 45:9–13. doi: 10.1093/jac/45.1.9. [DOI] [PubMed] [Google Scholar]
28.Kulkarni HM, Swamy CV, Jagannadham MV. 2014. Molecular characterization and functional analysis of outer membrane vesicles from the Antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions. J Proteome Res 13:1345–1358. doi: 10.1021/pr4009223. [DOI] [PubMed] [Google Scholar]
29.Park J, Kim M, Shin B, Kang M, Yang J, Lee TK, Park W. 2021. A novel decoy strategy for polymyxin resistance in Acinetobacter baumannii. Elife 10:e66988. doi: 10.7554/eLife.66988. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Strohmaier H, Remler P, Renner W, Högenauer G. 1995. Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12. J Bacteriol 177:4488–4500. doi: 10.1128/jb.177.15.4488-4500.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Deacon A, Ni Y, Coleman W, Jr, Ealick S. 2000. The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist. Structure 8:453–462. doi: 10.1016/S0969-2126(00)00128-3. [DOI] [PubMed] [Google Scholar]
32.Taylor PL, Blakely KM, De Leon GP, Walker JR, McArthur F, Evdokimova E, Zhang K, Valvano MA, Wright GD, Junop MS. 2008. Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants. J Biol Chem 283:2835–2845. doi: 10.1074/jbc.M706163200. [DOI] [PubMed] [Google Scholar]
33.Brooke JS, Valvano MA. 1996. Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase. J Biol Chem 271:3608–3614. doi: 10.1074/jbc.271.7.3608. [DOI] [PubMed] [Google Scholar]
34.Polissi A, Sperandeo P. 2014. The lipopolysaccharide export pathway in Escherichia coli: structure, organization and regulated assembly of the Lpt machinery. Marine Drugs 12:1023–1042. doi: 10.3390/md12021023. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Schmidt H, Hansen G, Singh S, Hanuszkiewicz A, Lindner B, Fukase K, Woodard RW, Holst O, Hilgenfeld R, Mamat U, Mesters JR. 2012. Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis. Proc Natl Acad Sci USA 109:6253–6258. doi: 10.1073/pnas.1119894109. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Wei J-R, Richie DL, Mostafavi M, Metzger LE, Rath CM, Sawyer WS, Takeoka KT, Dean CR. 2017. LpxK is essential for growth of Acinetobacter baumannii ATCC 19606: relationship to toxic accumulation of lipid A pathway intermediates. MSphere 2:e00199-17. doi: 10.1128/mSphere.00199-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Hicks G, Jia Z. 2018. Structural basis for the lipopolysaccharide export activity of the bacterial lipopolysaccharide transport system. Int J Mol Sci 19:2680. doi: 10.3390/ijms19092680. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Herrera CM, Hankins JV, Trent MS. 2010. Activation of PmrA inhibits LpxT-dependent phosphorylation of lipid A promoting resistance to antimicrobial peptides. Mol Microbiol 76:1444–1460. doi: 10.1111/j.1365-2958.2010.07150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Olaitan AO, Morand S, Rolain J-M. 2014. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 5:643. doi: 10.3389/fmicb.2014.00643. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Guo L, Lim KB, Poduje CM, Daniel M, Gunn JS, Hackett M, Miller SI. 1998. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95:189–198. doi: 10.1016/s0092-8674(00)81750-x. [DOI] [PubMed] [Google Scholar]
41.Band VI, Weiss DS. 2015. Mechanisms of antimicrobial peptide resistance in Gram-negative bacteria. Antibiotics (Basel) 4:18–41. doi: 10.3390/antibiotics4010018. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.McBroom AJ, Kuehn MJ. 2007. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63:545–558. doi: 10.1111/j.1365-2958.2006.05522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Jasim R, Baker MA, Zhu Y, Han M, Schneider-Futschik EK, Hussein M, Hoyer D, Li J, Velkov T. 2018. A comparative study of outer membrane proteome between paired colistin-susceptible and extremely colistin-resistant Klebsiella pneumoniae strains. ACS Infect Dis 4:1692–1704. doi: 10.1021/acsinfecdis.8b00174. [DOI] [PubMed] [Google Scholar]
44.Shankar-Sinha S, Valencia GA, Janes BK, Rosenberg JK, Whitfield C, Bender RA, Standiford TJ, Younger JG. 2004. The Klebsiella pneumoniae O antigen contributes to bacteremia and lethality during murine pneumonia. Infect Immun 72:1423–1430. doi: 10.1128/IAI.72.3.1423-1430.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Izquierdo L, Merino S, Regué M, Rodriguez F, Tomás JM. 2003. Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter. J Bacteriol 185:1634–1641. doi: 10.1128/JB.185.5.1634-1641.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Delgado MA, Mouslim C, Groisman EA. 2006. The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant. Mol Microbiol 60:39–50. doi: 10.1111/j.1365-2958.2006.05069.x. [DOI] [PubMed] [Google Scholar]
47.Chen Y-Y, Ko T-P, Lin C-H, Chen W-H, Wang AH-J. 2011. Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: a possible inhibition mechanism for the key enzyme in polymyxin resistance. J Struct Biol 175:300–310. doi: 10.1016/j.jsb.2011.04.010. [DOI] [PubMed] [Google Scholar]
48.Hung R-J, Chien H-S, Lin R-Z, Lin C-T, Vatsyayan J, Peng H-L, Chang H-Y. 2007. Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1. J Biol Chem 282:17738–17748. doi: 10.1074/jbc.M701824200. [DOI] [PubMed] [Google Scholar]
49.Álvarez D, Merino S, Tomás JM, Benedí VJ, Albertí S. 2000. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates. Infect Immun 68:953–955. doi: 10.1128/IAI.68.2.953-955.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Patro LPP, Rathinavelan T. 2019. Targeting the sugary armor of Klebsiella species. Front Cell Infect Microbiol 9:367. doi: 10.3389/fcimb.2019.00367. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Chen HD, Groisman EA. 2013. The biology of the PmrA/PmrB two-component system: the major regulator of lipopolysaccharide modifications. Annu Rev Microbiol 67:83–112. doi: 10.1146/annurev-micro-092412-155751. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Kox LF, Wösten MM, Groisman EA. 2000. A small protein that mediates the activation of a two-component system by another two-component system. EMBO J 19:1861–1872. doi: 10.1093/emboj/19.8.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Poirel L, Jayol A, Bontron S, Villegas M-V, Ozdamar M, Türkoglu S, Nordmann P. 2015. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 70:75–80. doi: 10.1093/jac/dku323. [DOI] [PubMed] [Google Scholar]
54.Miller AK, Brannon MK, Stevens L, Johansen HK, Selgrade SE, Miller SI, Høiby N, Moskowitz SM. 2011. PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients. Antimicrob Agents Chemother 55:5761–5769. doi: 10.1128/AAC.05391-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Nishino K, Senda Y, Yamaguchi A. 2008. CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes. J Antibiot (Tokyo) 61:120–127. doi: 10.1038/ja.2008.120. [DOI] [PubMed] [Google Scholar]
56.Otto K, Silhavy TJ. 2002. Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci USA 99:2287–2292. doi: 10.1073/pnas.042521699. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Li X-Z, Nikaido H. 2004. Efflux-mediated drug resistance in bacteria. Drugs 64:159–204. doi: 10.2165/00003495-200464020-00004. [DOI] [PubMed] [Google Scholar]
58.Joo H-S, Fu C-I, Otto M. 2016. Bacterial strategies of resistance to antimicrobial peptides. Philos Trans R Soc B 371:20150292. doi: 10.1098/rstb.2015.0292. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Kaparakis M, Turnbull L, Carneiro L, Firth S, Coleman HA, Parkington HC, Le Bourhis L, Karrar A, Viala J, Mak J, Hutton ML, Davies JK, Crack PJ, Hertzog PJ, Philpott DJ, Girardin SE, Whitchurch CB, Ferrero RL. 2010. Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cell Microbiol 12:372–385. doi: 10.1111/j.1462-5822.2009.01404.x. [DOI] [PubMed] [Google Scholar]
60.Lee M, Batuecas MT, Tomoshige S, Dominguez-Gil T, Mahasenan KV, Dik DA, Hesek D, Millan C, Uson I, Lastochkin E, Hermoso JA, Mobashery S. 2018. Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 115:4393–4398. doi: 10.1073/pnas.1801298115. [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Yakhnina AA, Bernhardt TG. 2020. The Tol-Pal system is required for peptidoglycan-cleaving enzymes to complete bacterial cell division. Proc Natl Acad Sci USA 117:6777–6783. doi: 10.1073/pnas.1919267117. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Rusch SL, Kendall DA. 2007. Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon. Biochim Biophys Acta 1768:5–12. doi: 10.1016/j.bbamem.2006.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Beckwith J. 2013. The Sec-dependent pathway. Res Microbiol 164:497–504. doi: 10.1016/j.resmic.2013.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Dalbey RE, Kuhn A. 2012. Protein traffic in Gram-negative bacteria—how exported and secreted proteins find their way. FEMS Microbiol Rev 36:1023–1045. doi: 10.1111/j.1574-6976.2012.00327.x. [DOI] [PubMed] [Google Scholar]
65.Auclair SM, Bhanu MK, Kendall DA. 2012. Signal peptidase I: cleaving the way to mature proteins. Protein Sci 21:13–25. doi: 10.1002/pro.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Lee J, Tomasek D, Santos TM, May MD, Meuskens I, Kahne D. 2019. Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA. Elife 8:e49787. doi: 10.7554/eLife.49787. [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Sklar JG, Wu T, Kahne D, Silhavy TJ. 2007. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev 21:2473–2484. doi: 10.1101/gad.1581007. [DOI] [PMC free article] [PubMed] [Google Scholar]
68.Carpousis AJ. 2007. The RNA degradosome of Escherichia coli: an mRNA-degrading machine assembled on RNase E. Annu Rev Microbiol 61:71–87. doi: 10.1146/annurev.micro.61.080706.093440. [DOI] [PubMed] [Google Scholar]
69.Truglio JJ, Croteau DL, Van Houten B, Kisker C. 2006. Prokaryotic nucleotide excision repair: the UvrABC system. Chem Rev 106:233–252. doi: 10.1021/cr040471u. [DOI] [PubMed] [Google Scholar]
70.Jiang Q, Chen J, Yang C, Yin Y, Yao K. 2019. Quorum sensing: a prospective therapeutic target for bacterial diseases. Biomed Res Int 2019:2015978. doi: 10.1155/2019/2015978. [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Osterman I, Dikhtyar YY, Bogdanov A, Dontsova O, Sergiev P. 2015. Regulation of flagellar gene expression in bacteria. Biochemistry (Mosc) 80:1447–1456. doi: 10.1134/S000629791511005X. [DOI] [PubMed] [Google Scholar]
72.Deris ZZ, Akter J, Sivanesan S, Roberts KD, Thompson PE, Nation RL, Li J, Velkov T. 2014. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity. J Antibiot (Tokyo) 67:147–151. doi: 10.1038/ja.2013.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
73.EUCAST. 2020. Clinical breakpoints: breakpoints and guidance. https://www.eucast.org/clinical_breakpoints/.
74.Weinstein MP. 2019. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute, Pittsburgh, PA. [Google Scholar]
75.The UniProt Consortium. 2017. UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169. doi: 10.1093/nar/gkw1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Kanehisa M, Goto S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30. doi: 10.1093/nar/28.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL. 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. doi: 10.1093/bioinformatics/btq249. [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Yu C-S, Cheng C-W, Su W-C, Chang K-C, Huang S-W, Hwang J-K, Lu C-H. 2014. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation. PLoS One 9:e99368. doi: 10.1371/journal.pone.0099368. [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Cox J, Mann M. 2008. MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372. doi: 10.1038/nbt.1511. [DOI] [PubMed] [Google Scholar]

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[B1] 1.Walsh TR, Weeks J, Livermore DM, Toleman MA. 2011. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infectious Diseases 11:355–362. doi: 10.1016/S1473-3099(11)70059-7. [DOI] [PubMed] [Google Scholar]

[B2] 2.Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N. 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infectious Diseases 10:597–602. doi: 10.1016/S1473-3099(10)70143-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. 2012. Ready for a world without antibiotics? The Pensières antibiotic resistance call to action. Antimicrob Resist Infect Control 14:11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Bialek-Davenet S, Criscuolo A, Ailloud F, Passet V, Jones L, Delannoy-Vieillard A-S, Garin B, Le Hello S, Arlet G, Nicolas-Chanoine M-H, Decré D, Brisse S. 2014. Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups. Emerg Infect Dis 20:1812–1820. doi: 10.3201/eid2011.140206. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Peerayeh SN, Rostami E, Siadat SD, Derakhshan S. 2014. High rate of aminoglycoside resistance in CTX-M-15 producing Klebsiella pneumoniae isolates in Tehran, Iran. Lab Med 45:231–237. doi: 10.1309/LMDQQW246NYAHHAD. [DOI] [PubMed] [Google Scholar]

[B6] 6.Endimiani A, Carias LL, Hujer AM, Bethel CR, Hujer KM, Perez F, Hutton RA, Fox WR, Hall GS, Jacobs MR, Paterson DL, Rice LB, Jenkins SG, Tenover FC, Bonomo RA. 2008. Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrob Agents Chemother 52:2680–2682. doi: 10.1128/AAC.00158-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Velkov T, Roberts KD, Nation RL, Thompson PE, Li J. 2013. Pharmacology of polymyxins: new insights into an “old” class of antibiotics. Future Microbiol 8:711–724. doi: 10.2217/fmb.13.39. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Gales AC, Jones R, Sader HS. 2006. Global assessment of the antimicrobial activity of polymyxin B against 54 731 clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme (2001–2004). Clin Microbiol Infect 12:315–321. doi: 10.1111/j.1469-0691.2005.01351.x. [DOI] [PubMed] [Google Scholar]

[B9] 9.Weterings V, Zhou K, Rossen J, van Stenis D, Thewessen E, Kluytmans J, Veenemans J. 2015. An outbreak of colistin-resistant Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae in the Netherlands (July to December 2013), with inter-institutional spread. Eur J Clin Microbiol Infect Dis 34:1647–1655. doi: 10.1007/s10096-015-2401-2. [DOI] [PubMed] [Google Scholar]

[B10] 10.Bratu S, Tolaney P, Karumudi U, Quale J, Mooty M, Nichani S, Landman D. 2005. Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents. J Antimicrob Chemother 56:128–132. doi: 10.1093/jac/dki175. [DOI] [PubMed] [Google Scholar]

[B11] 11.Elemam A, Rahimian J, Mandell W. 2009. Infection with panresistant Klebsiella pneumoniae: a report of 2 cases and a brief review of the literature. Clin Infect Dis 49:271–274. doi: 10.1086/600042. [DOI] [PubMed] [Google Scholar]

[B12] 12.Hussein M, Wong LJM, Zhao J, Rees VE, Allobawi R, Sharma R, Rao GG, Baker M, Li J, Velkov T. 2022. Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant Klebsiella pneumoniae. Comput Struct Biotechnol J 20:1077–1087. doi: 10.1016/j.csbj.2022.02.021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Hussein M, Hu X, Paulin OK, Crawford S, Zhou QT, Baker M, Schneider-Futschik EK, Zhu Y, Li J, Velkov T. 2020. Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens. Comput Struct Biotechnol J 18:2247–2258. doi: 10.1016/j.csbj.2020.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Hsu Chen CC, Feingold DS. 1973. Mechanism of polymyxin B action and selectivity toward biologic membranes. Biochemistry 12:2105–2111. doi: 10.1021/bi00735a014. [DOI] [PubMed] [Google Scholar]

[B15] 15.Falagas ME, Rafailidis PI, Matthaiou DK. 2010. Resistance to polymyxins: mechanisms, frequency and treatment options. Drug Resist Updat 13:132–138. doi: 10.1016/j.drup.2010.05.002. [DOI] [PubMed] [Google Scholar]

[B16] 16.Raetz CR, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 76:295–329. doi: 10.1146/annurev.biochem.76.010307.145803. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Cheng H-Y, Chen Y-F, Peng H-L. 2010. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci 17:60. doi: 10.1186/1423-0127-17-60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Clements A, Tull D, Jenney AW, Farn JL, Kim S-H, Bishop RE, McPhee JB, Hancock REW, Hartland EL, Pearse MJ, Wijburg OLC, Jackson DC, McConville MJ, Strugnell RA. 2007. Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides. J Biol Chem 282:15569–15577. doi: 10.1074/jbc.M701454200. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Velkov T, Soon RL, Chong PL, Huang JX, Cooper MA, Azad MAK, Baker MA, Thompson PE, Roberts K, Nation RL, Clements A, Strugnell RA, Li J. 2013. Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A. Innate Immun 19:265–277. doi: 10.1177/1753425912459092. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Llobet E, March C, Giménez P, Bengoechea JA. 2009. Klebsiella pneumoniae OmpA confers resistance to antimicrobial peptides. Antimicrob Agents Chemother 53:298–302. doi: 10.1128/AAC.00657-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Schwechheimer C, Kuehn MJ. 2015. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13:605–619. doi: 10.1038/nrmicro3525. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Horstman AL, Kuehn MJ. 2000. Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem 275:12489–12496. doi: 10.1074/jbc.275.17.12489. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Dorward DW, Garon CF, Judd RC. 1989. Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol 171:2499–2505. doi: 10.1128/jb.171.5.2499-2505.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Lee E-Y, Bang JY, Park GW, Choi D-S, Kang JS, Kim H-J, Park K-S, Lee J-O, Kim Y-K, Kwon K-H, Kim K-P, Gho YS. 2007. Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli. Proteomics 7:3143–3153. doi: 10.1002/pmic.200700196. [DOI] [PubMed] [Google Scholar]

[B25] 25.Dell’Annunziata F, Dell’Aversana C, Doti N, Donadio G, Dal Piaz F, Izzo V, De Filippis A, Galdiero M, Altucci L, Boccia G, Galdiero M, Folliero V, Franci G. 2021. Outer membrane vesicles derived from Klebsiella pneumoniae are a driving force for horizontal gene transfer. Int J Mol Sci 22:8732. doi: 10.3390/ijms22168732. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Manning AJ, Kuehn MJ. 2011. Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol 11:258. doi: 10.1186/1471-2180-11-258. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Ciofu O, Beveridge TJ, Kadurugamuwa J, Walther-Rasmussen J, Høiby N. 2000. Chromosomal β-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. J Antimicrob Chemother 45:9–13. doi: 10.1093/jac/45.1.9. [DOI] [PubMed] [Google Scholar]

[B28] 28.Kulkarni HM, Swamy CV, Jagannadham MV. 2014. Molecular characterization and functional analysis of outer membrane vesicles from the Antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions. J Proteome Res 13:1345–1358. doi: 10.1021/pr4009223. [DOI] [PubMed] [Google Scholar]

[B29] 29.Park J, Kim M, Shin B, Kang M, Yang J, Lee TK, Park W. 2021. A novel decoy strategy for polymyxin resistance in Acinetobacter baumannii. Elife 10:e66988. doi: 10.7554/eLife.66988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Strohmaier H, Remler P, Renner W, Högenauer G. 1995. Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12. J Bacteriol 177:4488–4500. doi: 10.1128/jb.177.15.4488-4500.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Deacon A, Ni Y, Coleman W, Jr, Ealick S. 2000. The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist. Structure 8:453–462. doi: 10.1016/S0969-2126(00)00128-3. [DOI] [PubMed] [Google Scholar]

[B32] 32.Taylor PL, Blakely KM, De Leon GP, Walker JR, McArthur F, Evdokimova E, Zhang K, Valvano MA, Wright GD, Junop MS. 2008. Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants. J Biol Chem 283:2835–2845. doi: 10.1074/jbc.M706163200. [DOI] [PubMed] [Google Scholar]

[B33] 33.Brooke JS, Valvano MA. 1996. Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase. J Biol Chem 271:3608–3614. doi: 10.1074/jbc.271.7.3608. [DOI] [PubMed] [Google Scholar]

[B34] 34.Polissi A, Sperandeo P. 2014. The lipopolysaccharide export pathway in Escherichia coli: structure, organization and regulated assembly of the Lpt machinery. Marine Drugs 12:1023–1042. doi: 10.3390/md12021023. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Schmidt H, Hansen G, Singh S, Hanuszkiewicz A, Lindner B, Fukase K, Woodard RW, Holst O, Hilgenfeld R, Mamat U, Mesters JR. 2012. Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis. Proc Natl Acad Sci USA 109:6253–6258. doi: 10.1073/pnas.1119894109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Wei J-R, Richie DL, Mostafavi M, Metzger LE, Rath CM, Sawyer WS, Takeoka KT, Dean CR. 2017. LpxK is essential for growth of Acinetobacter baumannii ATCC 19606: relationship to toxic accumulation of lipid A pathway intermediates. MSphere 2:e00199-17. doi: 10.1128/mSphere.00199-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37.Hicks G, Jia Z. 2018. Structural basis for the lipopolysaccharide export activity of the bacterial lipopolysaccharide transport system. Int J Mol Sci 19:2680. doi: 10.3390/ijms19092680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Herrera CM, Hankins JV, Trent MS. 2010. Activation of PmrA inhibits LpxT-dependent phosphorylation of lipid A promoting resistance to antimicrobial peptides. Mol Microbiol 76:1444–1460. doi: 10.1111/j.1365-2958.2010.07150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39.Olaitan AO, Morand S, Rolain J-M. 2014. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 5:643. doi: 10.3389/fmicb.2014.00643. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40.Guo L, Lim KB, Poduje CM, Daniel M, Gunn JS, Hackett M, Miller SI. 1998. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95:189–198. doi: 10.1016/s0092-8674(00)81750-x. [DOI] [PubMed] [Google Scholar]

[B41] 41.Band VI, Weiss DS. 2015. Mechanisms of antimicrobial peptide resistance in Gram-negative bacteria. Antibiotics (Basel) 4:18–41. doi: 10.3390/antibiotics4010018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.McBroom AJ, Kuehn MJ. 2007. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63:545–558. doi: 10.1111/j.1365-2958.2006.05522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Jasim R, Baker MA, Zhu Y, Han M, Schneider-Futschik EK, Hussein M, Hoyer D, Li J, Velkov T. 2018. A comparative study of outer membrane proteome between paired colistin-susceptible and extremely colistin-resistant Klebsiella pneumoniae strains. ACS Infect Dis 4:1692–1704. doi: 10.1021/acsinfecdis.8b00174. [DOI] [PubMed] [Google Scholar]

[B44] 44.Shankar-Sinha S, Valencia GA, Janes BK, Rosenberg JK, Whitfield C, Bender RA, Standiford TJ, Younger JG. 2004. The Klebsiella pneumoniae O antigen contributes to bacteremia and lethality during murine pneumonia. Infect Immun 72:1423–1430. doi: 10.1128/IAI.72.3.1423-1430.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45.Izquierdo L, Merino S, Regué M, Rodriguez F, Tomás JM. 2003. Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter. J Bacteriol 185:1634–1641. doi: 10.1128/JB.185.5.1634-1641.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46.Delgado MA, Mouslim C, Groisman EA. 2006. The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant. Mol Microbiol 60:39–50. doi: 10.1111/j.1365-2958.2006.05069.x. [DOI] [PubMed] [Google Scholar]

[B47] 47.Chen Y-Y, Ko T-P, Lin C-H, Chen W-H, Wang AH-J. 2011. Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: a possible inhibition mechanism for the key enzyme in polymyxin resistance. J Struct Biol 175:300–310. doi: 10.1016/j.jsb.2011.04.010. [DOI] [PubMed] [Google Scholar]

[B48] 48.Hung R-J, Chien H-S, Lin R-Z, Lin C-T, Vatsyayan J, Peng H-L, Chang H-Y. 2007. Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1. J Biol Chem 282:17738–17748. doi: 10.1074/jbc.M701824200. [DOI] [PubMed] [Google Scholar]

[B49] 49.Álvarez D, Merino S, Tomás JM, Benedí VJ, Albertí S. 2000. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates. Infect Immun 68:953–955. doi: 10.1128/IAI.68.2.953-955.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50.Patro LPP, Rathinavelan T. 2019. Targeting the sugary armor of Klebsiella species. Front Cell Infect Microbiol 9:367. doi: 10.3389/fcimb.2019.00367. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51.Chen HD, Groisman EA. 2013. The biology of the PmrA/PmrB two-component system: the major regulator of lipopolysaccharide modifications. Annu Rev Microbiol 67:83–112. doi: 10.1146/annurev-micro-092412-155751. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52.Kox LF, Wösten MM, Groisman EA. 2000. A small protein that mediates the activation of a two-component system by another two-component system. EMBO J 19:1861–1872. doi: 10.1093/emboj/19.8.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53.Poirel L, Jayol A, Bontron S, Villegas M-V, Ozdamar M, Türkoglu S, Nordmann P. 2015. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 70:75–80. doi: 10.1093/jac/dku323. [DOI] [PubMed] [Google Scholar]

[B54] 54.Miller AK, Brannon MK, Stevens L, Johansen HK, Selgrade SE, Miller SI, Høiby N, Moskowitz SM. 2011. PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients. Antimicrob Agents Chemother 55:5761–5769. doi: 10.1128/AAC.05391-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55.Nishino K, Senda Y, Yamaguchi A. 2008. CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes. J Antibiot (Tokyo) 61:120–127. doi: 10.1038/ja.2008.120. [DOI] [PubMed] [Google Scholar]

[B56] 56.Otto K, Silhavy TJ. 2002. Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci USA 99:2287–2292. doi: 10.1073/pnas.042521699. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57.Li X-Z, Nikaido H. 2004. Efflux-mediated drug resistance in bacteria. Drugs 64:159–204. doi: 10.2165/00003495-200464020-00004. [DOI] [PubMed] [Google Scholar]

[B58] 58.Joo H-S, Fu C-I, Otto M. 2016. Bacterial strategies of resistance to antimicrobial peptides. Philos Trans R Soc B 371:20150292. doi: 10.1098/rstb.2015.0292. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B59] 59.Kaparakis M, Turnbull L, Carneiro L, Firth S, Coleman HA, Parkington HC, Le Bourhis L, Karrar A, Viala J, Mak J, Hutton ML, Davies JK, Crack PJ, Hertzog PJ, Philpott DJ, Girardin SE, Whitchurch CB, Ferrero RL. 2010. Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cell Microbiol 12:372–385. doi: 10.1111/j.1462-5822.2009.01404.x. [DOI] [PubMed] [Google Scholar]

[B60] 60.Lee M, Batuecas MT, Tomoshige S, Dominguez-Gil T, Mahasenan KV, Dik DA, Hesek D, Millan C, Uson I, Lastochkin E, Hermoso JA, Mobashery S. 2018. Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 115:4393–4398. doi: 10.1073/pnas.1801298115. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B61] 61.Yakhnina AA, Bernhardt TG. 2020. The Tol-Pal system is required for peptidoglycan-cleaving enzymes to complete bacterial cell division. Proc Natl Acad Sci USA 117:6777–6783. doi: 10.1073/pnas.1919267117. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B62] 62.Rusch SL, Kendall DA. 2007. Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon. Biochim Biophys Acta 1768:5–12. doi: 10.1016/j.bbamem.2006.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B63] 63.Beckwith J. 2013. The Sec-dependent pathway. Res Microbiol 164:497–504. doi: 10.1016/j.resmic.2013.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B64] 64.Dalbey RE, Kuhn A. 2012. Protein traffic in Gram-negative bacteria—how exported and secreted proteins find their way. FEMS Microbiol Rev 36:1023–1045. doi: 10.1111/j.1574-6976.2012.00327.x. [DOI] [PubMed] [Google Scholar]

[B65] 65.Auclair SM, Bhanu MK, Kendall DA. 2012. Signal peptidase I: cleaving the way to mature proteins. Protein Sci 21:13–25. doi: 10.1002/pro.757. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B66] 66.Lee J, Tomasek D, Santos TM, May MD, Meuskens I, Kahne D. 2019. Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA. Elife 8:e49787. doi: 10.7554/eLife.49787. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B67] 67.Sklar JG, Wu T, Kahne D, Silhavy TJ. 2007. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev 21:2473–2484. doi: 10.1101/gad.1581007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B68] 68.Carpousis AJ. 2007. The RNA degradosome of Escherichia coli: an mRNA-degrading machine assembled on RNase E. Annu Rev Microbiol 61:71–87. doi: 10.1146/annurev.micro.61.080706.093440. [DOI] [PubMed] [Google Scholar]

[B69] 69.Truglio JJ, Croteau DL, Van Houten B, Kisker C. 2006. Prokaryotic nucleotide excision repair: the UvrABC system. Chem Rev 106:233–252. doi: 10.1021/cr040471u. [DOI] [PubMed] [Google Scholar]

[B70] 70.Jiang Q, Chen J, Yang C, Yin Y, Yao K. 2019. Quorum sensing: a prospective therapeutic target for bacterial diseases. Biomed Res Int 2019:2015978. doi: 10.1155/2019/2015978. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B71] 71.Osterman I, Dikhtyar YY, Bogdanov A, Dontsova O, Sergiev P. 2015. Regulation of flagellar gene expression in bacteria. Biochemistry (Mosc) 80:1447–1456. doi: 10.1134/S000629791511005X. [DOI] [PubMed] [Google Scholar]

[B72] 72.Deris ZZ, Akter J, Sivanesan S, Roberts KD, Thompson PE, Nation RL, Li J, Velkov T. 2014. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity. J Antibiot (Tokyo) 67:147–151. doi: 10.1038/ja.2013.111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B73] 73.EUCAST. 2020. Clinical breakpoints: breakpoints and guidance. https://www.eucast.org/clinical_breakpoints/.

[B74] 74.Weinstein MP. 2019. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute, Pittsburgh, PA. [Google Scholar]

[B75] 75.The UniProt Consortium. 2017. UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169. doi: 10.1093/nar/gkw1099. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B76] 76.Kanehisa M, Goto S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30. doi: 10.1093/nar/28.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B77] 77.Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL. 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. doi: 10.1093/bioinformatics/btq249. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B78] 78.Yu C-S, Cheng C-W, Su W-C, Chang K-C, Huang S-W, Hwang J-K, Lu C-H. 2014. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation. PLoS One 9:e99368. doi: 10.1371/journal.pone.0099368. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B79] 79.Cox J, Mann M. 2008. MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372. doi: 10.1038/nbt.1511. [DOI] [PubMed] [Google Scholar]

PERMALINK

Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae

Maytham Hussein

Raad Jasim

Hakan Gocol

Mark Baker

Varsha J Thombare

James Ziogas

Aayush Purohit

Gauri G Rao

Jian Li

Tony Velkov

Roles

ABSTRACT

INTRODUCTION

RESULTS AND DISCUSSION

OMV subproteome characterization.

FIG 1.

TABLE 1.

Lipopolysaccharide biosynthesis and modification.

FIG 2.

O-antigen nucleotide sugar biosynthesis.

Two-component systems and cationic antimicrobial peptide (CAMPs) resistance.

β-lactam resistance.

Peptidoglycan biosynthesis.

FIG 3.

Protein export and outer membrane proteins (OMPs) complex biosynthesis.

FIG 4.

RNA degradation and nucleotide excision/repair.

Quorum sensing (QS).

Conclusions.

MATERIALS AND METHODS

Reagents.

Bacterial isolates and growth conditions.

Measurements of MICs.

Isolation of outer membrane vesicles (OMVs).

Ultra-high-performance liquid chromatography coupled with mass spectrometry.

Bioinformatics analysis.

ACKNOWLEDGMENTS

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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