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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Jan 30;1848(11 0 0):3021–3025. doi: 10.1016/j.bbamem.2015.01.013

S. Typhimurium strategies to resist killing by cationic antimicrobial peptides

Susana Matamouros 1, Samuel I Miller 1,2,3
PMCID: PMC4520786  NIHMSID: NIHMS664260  PMID: 25644871

Abstract

S.Typhimurium is a broad host range Gram-negative patho that must evade killing by host innate immune systems to colonize, replicate, cause disease, and be transmitted to other hosts. A major pathogenic strategy of Salmonellae is entrance, survival, and replication within eukaryotic cell phagocytic vacuoles. These phagocytic vacuoles and gastrointestinal mucosal surfaces contain multiple cationic antimicrobial peptides (CAMPs) which control invading bacteria. S. Typhimurium possesses several key mechanisms to resist killing by CAMPs which involve sensing CAMPs and membrane damage to activate signaling cascades that result in remodeling of the bacterial envelope to reduce its overall negative charge with an increase in hydrophobicity to decrease binding and effectiveness of CAMPs. Moreover Salmonellae have additional mechanisms to resist killing by CAMPs including an outer membrane protease which targets cationic peptides at the surface, and specific efflux pumps which protect the inner membrane from damage.

Keywords: Salmonella Typhimurium, Resistance to cationic antimicrobial peptides, Polymyxin, outer-membrane remodeling, lipid A modifications

Introduction

Antimicrobial peptides (AMPs) are ubiquitously produced as part of mammalian innate immune systems and make up the entire immune system of insects and many non-vertebrate animals. They are structurally diverse small amphipathic peptides in which a cationic and a hydrophobic surface facilitate interaction with biological membranes [1–4]. Unlike eukaryotic cells, the bacterial outer layer net surface charge is anionic [2]. Gram-negative bacteria have negatively-charged lipopolysaccharides (LPS) as the major constituent of the outer-leaflet of their outer-membranes and Gram-positive bacteria have acidic polysaccharides (teichoic and teichuronic acids) decorating their surface. Consequently most AMPs with activity against bacteria are cationic which allows for binding to the negatively charged bacterial surface [2–4]. Thus, surface remodeling aimed at decreasing the negative charge and increasing hydrophobicity of bacterial membranes is often employed as a first line of defense in bacterial resistance to antimicrobial peptides. Remodeling of the bacterial surface has been extensively studied in the Gram-negative pathogenic bacterium Salmonella enterica serovar Typhimurium. Salmonella is able to reduce recognition by the host innate immune system and binding of CAMPs to its surface by specific modification of its outer membrane components. Several strategies have been identified in Salmonellae and other Gram-negative bacteria that result in the modification of the LPS structure and properties as well as sequestering, efflux and proteolytic degradation of AMPs.

Regulatory systems

Salmonella possesses several regulatory systems that control modifications necessary for AMP resistance. The regulation of the proteins involved in these processes is complex and involves several regulatory proteins such as the alternative sigma factor, RpoE [5] and several two-component systems: PhoPQ, PmrAB and RcsABCDF systems (Figure 1) [6–8]. S. Typhimurium and other Gram-negative bacteria regulate most changes to the outer-membrane through the two-component system PhoPQ [9–11]. PhoQ is an inner-membrane histidine kinase that is activated once Salmonella is inside host cells and responds to low pH and the presence of AMPs [7, 12–14]. The presence of these signals leads to the phosphorylation of the cytoplasmic response regulator PhoP (Figure 1). PhoQ periplasmic domain contains a negatively charged surface (acidic patch) facing the membrane that is tethered to the anionic phospholipid head-groups of the inner-membrane by the presence of metal ions such as Ca2+ and Mg2+ keeping it in a repressed state [15]. Direct interaction of the PhoQ periplasmic domain acidic patch with the positively charged surface of CAMPs disrupts the metal bridges and induces conformational changes in PhoQ that lead to PhoQ autophosphorylation and subsequent transfer of the phosphate group to PhoP (Figure 1) [13]. Once phosphorylated, PhoP controls the expression of several genes involved in resistance to AMPs and intracellular survival and hence virulence.

Figure 1.

Figure 1

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Regulatory systems that control several modifications essential for Salmonella resistance to CAMPs. Upon exposure to CAMPs there is disruption of the outer membrane barrier. PhoQ is able to directly bind CAMPs via its periplasmic domain which results in activation of the PhoPQ and the PmrAB operons. These operons encode several proteins essential for resistance to CAMPs, among which is the outer-membrane protease PgtE. Activation of the Rcs phosphorelay system occurs via the outer-membrane lipoprotein RcsF that senses membrane damage caused by CAMPs. The concerted activation of these regulons results in increased resistance to CAMPs.

PhoPQ also regulates the transcription of another two-component system, PmrAB that is necessary for Salmonella resistance to AMPs [6, 16]. Upon activation PhoP induced synthesis of PmrD prevents the dephosphorylation of PmrA keeping it in an activating state (Figure 1) [17, 18]. Activation of PmrAB can also occur in the presence of ferric iron or low pH [19, 20]. PmrA directly controls the expression of several genes involved in LPS modifications that are absolutely necessary for resistance to Polymyxin B and other CAMPs.

The Rcs regulatory system involves several regulatory proteins and a multiple-step phosphotransfer cascade. It is composed of two inner-membrane sensors, the RcsC hybrid kinase and the RcsD histidine phosphotransfer protein, an outer membrane inner leaflet and periplasmic lipoprotein RcsF and two cytoplasmic regulatory proteins, RcsA and RcsB (Figure 1) [21, 22]. RcsF is essential for the activation of this system in the presence of CAMP, likely through specific disorder of the outer membrane and alteration in the localization or conformation of RcsF [8]. The Rcs system controls the expression of the capsular polysaccharide genes as well as other genes necessary for Salmonella resistance to antimicrobial peptides and persistence in mice [23, 24].

Regulation of O-antigen length

The outer-leaflet of the outer-membrane of Gram-negative bacteria is mainly composed of LPS. LPS is a complex amphiphilic molecule composed of three distinct domains: the hypervariable and highly immunogenic O-region formed by repeating oligosaccharide subunits; a short core oligosaccharide region usually common to all members of a bacterial genus and; a membrane anchoring hydrophobic lipid composed of a phosphorylated glucosamine disaccharide that in enteric bacteria most often has 6 attached fatty acids, termed lipid A. Since LPS is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, it is therefore a first barrier in the defense against external toxins such as AMPs. Being the outermost portion of the LPS molecule and bacterial cellular surface, the O-antigen serves as a protective barrier from CAMPs and other membrane active compounds. It hides the negatively charged phosphate groups of the core and lipid A inhibiting the electrostatic attraction between the CAMPs and these two domains of the LPS molecule. S. Typhimurium can produce short LPS species with the O-antigen domain containing between 1 and 15 oligosaccharide repeat units, long LPS species that have 16 to 35 O-antigen repeat units [25], or very long LPS chains can have over 100 repeat units [26]. The biosynthesis of the short, long and very long O-antigen species is controlled by the length regulators: PbgE2,3, WzzST and FepE, respectively. The expression of these proteins is under the direct control of the PmrAB regulatory system [27, 28]. The presence of O-antigen has been shown to be important to resistance to Polymyxin B since a S. Typhimurium mutant devoid of O-antigen shows high susceptibility to this antimicrobial peptide [29]. However, S. Typhimurium mutants devoid of long and/or very long O-antigen species show only a mild increase in susceptibility to Polymyxin B [27, 29] and the LPS of PhoP constitutive strains present shorter O-antigen chains [30, 31]. Together this suggests that short O-antigen chains are sufficient in providing a barrier against Polymyxin B and possibly other antimicrobial peptides. It is conceivable that the overall shorter length leads to a more uniform surface, rather than a number of shorter and longer chains, which could form a more effective barrier.

Reduction of the anionic character of the bacterial surface

Another strategy employed by bacteria to resist AMPs is to reduce the negative charge of its surface. In Gram-negative this is achieved by modification of the anionic phosphate groups in the core and lipid A regions of the LPS with the addition of cationic aminoarabinose and zwitterionic phosphoethanolamine moieties (Figure 2) [30, 32, 33]. The lipid A of Salmonella is phosphorylated at the 1 and 4′ positions [34]. The addition of aminoarabinose and phosphoethanolamine at these positions is catalyzed by the PmrA-dependent ArnT (PmrK) [35, 36] and EptA (PmrC) enzymes [37], respectively. In S. Typhimurium, aminoarabinose is usually added to the 4′-phosphate group and the phosphoethanolamine group added to the 1-phosphate group of lipid A, however in some cases both positions can be modified by the same cationic group [38]. PmrA activation also inhibits the activity of LpxT, an undecaprenyl phosphotransferase responsible for increasing the negative charge of LPS by adding an additional phosphate at the 1-position [39–41]. The concerted inhibition of LpxT and the addition of cationic groups to lipid A highly contribute to S. Typhimurium resistance to Polymyxin B and other CAMPs [37, 39–42] by decreasing the negative charge of the outer membrane and therefore electrostactic interactions with CAMPs.

Figure 2.

Figure 2

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S. Typhimurium LPS structure. Important modifications for resistance to CAMPs are indicated in red along with the enzyme that catalyzes the reaction. The addition of cationic aminoarabinose and zwitterionic phosphoethanolamine moieties neutralizes the negative charges of the phosphate groups. Lipid A palmitoylation is catalyzed by PagP and results in increased membrane hydrophobicity and thickness which along with the charge neutralization help prevent or delay CAMPs insertion in the membrane.

Decrease in bacterial surface membrane fluidity

In order to diminish the interaction and uptake of AMPs Salmonella species are able to reduce the fluidity of their outer membrane by increasing its hydrophobicity. This is achieved by adding additional palmitate to Lipid A to form hepta-acylated Lipid A. This modification is catalyzed by the outer-membrane enzyme PagP that catalyzes lipid A palmitoylation (Figure 2) [43, 44]. The expression of this enzyme is under the control of the PhoPQ two component system and its activity highly contributes to CAMPs resistance [43, 44]. Recently, we have demonstrated that PagP can also catalyze the palmitoylation of phosphatidylglycerol on the outer-membrane of S. Typhimurium [45]. In certain S. Typhimurium mutants [46], possibly upon disruption of the outer-membrane barrier, phospholipids migrate to the outer-leaflet of the outer-membrane to try to keep membrane integrity providing an additional substrate for PagP. Palmitoylation of Lipid A and phosphatidylglycerol contribute to the increase of the hydrophobic moment and thickness of the outer membrane, which are thought to decrease fluidity and prevent and delay CAMP insertion.

Binding and Eflux of CAMPs

Additional resistance mechanisms employed by Salmonella and other bacteria include the binding, active efflux, or proteolytic degradation of AMPs before they can exert their action in their intra- or extra-cellular targets.

Two S. Typhimurium PhoPQ regulated proteins, Mig-14 and VirK are necessary for resistance to the cathelicidin-related anti-microbial peptide (CRAMP) and Polymyxin B [23, 47–49]. These two adjacent genes are transcriptionally upregulated once inside macrophages and in the presence of AMPs [49]. Mutants in these genes are attenuated in macrophage survival and long-term persistence in mice though their mechanism of action or alteration of the cell envelope is unknown [48, 50]. It has been shown that the inner membrane proteins, Mig-14 and VirK inhibit binding of CRAMP to S. Typhimurium cells and therefore it was proposed that by binding CRAMP they prevent access of this AMP to its cytoplasmic target [47]. Another hypothesis is that these proteins could interact with efflux or degradations systems.

The only efflux systems identified in Salmonella for the export of AMPs are encoded by the sap (sensitivity to antimicrobial peptides) genes [51–53]. These genes are necessary for CAMP resistance and for virulence in a mouse model of infection [51]. The sapABCDF operon was shown to encode an ABC transporter that is involved in resistance to small cationic peptides [52] while sapG and sapJ are involved in peptide resistance and potassium transport and could make part of the SapABCDF complex [53].

Proteolytic degradation of CAMPs

In S. Typhimurium proteolytic degradation of CAMPs is accomplished by the outer-membrane protease PgtE [54] from the OmpT/Pla family of outer-membrane proteases (Figure 1). Although transcription of PgtE was found to be PhoPQ independent its localization and or stability within the outer-membrane is dependent upon PhoPQ activation and its activity was found to be directed specifically to alpha-helical peptides such as C18G and LL-37 [54]. PgtE does not confer resistance to β-sheet AMPs which form more rigid structures stabilized by disulfide bonds. Some of these AMPs such as α- and β-defensins only exist in mammals and may have evolved as part of the mammalian defense response to microbial invasions to overcome degradation by proteases produced by several bacteria [55].

Conclusion

Salmonellae are intracellular pathogens which through the course of infection encounter a variety of innate immune CAMPs with diverse targets and modes of action. S. Typhimurium possesses multiple strategies to resist killing by CAMPs. All these processes are highly regulated and often redundant, similar to mammalian innate immune receptors and responses. Inducible AMP resistance is an old process in bacteria since AMPs are produced by bacteria and may have originally evolved before eukaryotes as a mechanism to resist polymyxins produced by Bacillus spp. Resistance to AMPs likely facilitated the success of Salmonellae as gastrointestinal and systemic intracellular pathogens. Understanding these mechanisms and others involved in the inducible response to AMPs that result in an increase in outer membrane barrier could promote the development of new antibiotics that target this barrier, improve the action of current antibiotics which are ineffective as a result of this inducible barrier and may lead to the identification of new antibiotic targets for antimicrobial development.

Highlights.

  • An effective outer membrane barrier is essential for evading the host immune system

  • Multiple regulatory systems control the remodel of the bacterial surface

  • Decreased negative charge prevents CAMP from binding to the bacterial surface

  • Proteolytic degradation and efflux systems inhibit CAMP from reaching its targets

Acknowledgments

This work was funded by the NIH Grant R01AI030479 (to S.I.M.).

Abbreviations

AMP

Antimicrobial peptides

CAMP

Cationic antimicrobial peptides

LPS

Lipopolysaccharide

CRAMP

cathelicidin-related anti-microbial peptide

Footnotes

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