Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production

Yi-Tzu Lee; Te-Li Chen; Shu-Chen Kuo; Yu-Han Liu; Meng-Chang Lee; Ming-Fen Chuang; Mei-Chei Tan; I-Ming Lee; Ya-Sung Yang; Yung-Chih Wang

doi:10.1371/journal.ppat.1014364

. 2026 Jul 14;22(7):e1014364. doi: 10.1371/journal.ppat.1014364

Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production

Yi-Tzu Lee ^1,^2,^#, Te-Li Chen ^3,^#, Shu-Chen Kuo ⁴, Yu-Han Liu ⁵, Meng-Chang Lee ⁶, Ming-Fen Chuang ⁵, Mei-Chei Tan ⁴, I-Ming Lee ^7,⁸, Ya-Sung Yang ⁵, Yung-Chih Wang ^5,^*

Editor: Mario F Feldman⁹

PMCID: PMC13367702 PMID: 42447074

Abstract

Bacterial strains harboring lipooligosaccharides (LOS) with a core region are more pathogenic than those without it; however, the underlying mechanism remains to be fully elucidated. Lipid A has been believed to be the primary moiety of LOS that regulates Toll-like receptor 4 (TLR4) signaling, with the core region having minimal impact. In this study, we aimed to elucidate the influence of the LOS core region of Acinetobacter baumannii on immune response and pathogenesis. We conducted a series of step-by-step experiments and deciphered the association between the core region of the LOS and the immune response. The influence of this core region on the mechanism of TLR4 signaling pathways and further pathogenesis has also been deciphered. One A. baumannii wild strain with an intact LOS core region, its LOS core region gene (lpsB)-deficient strain, and the lpsB gene-complemented strain were used in this study. Membrane vesicles released from the strains were collected, quantified, normalized to the particle number, and used in subsequent experiments. Cytokine and chemokine gene expression, protein production, and RNA sequencing analyses were measured. A step-by-step approach was used to explore the underlying mechanism. The effect of the LOS core region on pathogenesis was determined using mouse experiments. Specifically, the LOS core promotes CD14-associated TLR4 endocytosis and boosting the expression of TRIF-associated genes, including Infb1. This enhancement in interferon-β production correlates with increased pathogenicity in an animal model. These findings highlight the significance of the LOS core region in modulating immune responses through TRIF signaling, challenging previous assumptions regarding the role of this core region in LOS-mediated pathogenesis.

Author summary

In this study, following a series of experiments, we determined that the core region of lipooligosaccharides (LOS) enhanced CD14-dependent toll-like receptor 4 (TLR4) endocytosis. It further enhanced interferon-β (IFN-β) production through the TLR4-TRIF signaling pathway and contributed to higher pathogenicity. Initially, we found that an A. baumannii strain harboring the core region of LOS displayed higher virulence than a strain without this core region. We further showed that the membrane vesicles (MVs) released from A. baumannii with intact core stimulated greater IFN-β production and higher Infb1 gene expression than those released from A. baumannii without intact core. The core region of LOS upregulated the expression of IFN-β-associated genes, as revealed from the RNA sequencing analysis. We further confirmed that IFN-β production was stimulated through the CD14-TLR4-TRIF signaling pathway. In animal experiments, IFNAR1-deficient mice (Ifnar1-/-) exhibited a higher survival rate than did the wild-type mice after challenge with MVs containing an intact LOS core. Treatment with anti-IFN-β antibodies, but not isotype control antibodies, significantly improved the survival of mice exposed to MVs released from A. baumannii. We believe that our study makes a significant contribution because it demonstrates that the core region of LOS promotes CD14-associated TLR4 endocytosis, thereby increasing the expression of TRIF-associated genes, including Infb1, and further enhances the pathogenesis of A. baumannii.

Introduction

Acinetobacter baumannii is a leading pathogen of nosocomial infections [1,2], and is notorious for its rapid evolution of resistance to numerous antimicrobial agents [3,4]. Despite extensive studies on resistance mechanisms, the pathogenesis of A. baumannii infection has not been sufficiently studied [3,5]. In gram-negative bacteria, lipopolysaccharide (LPS) is a major virulence factor comprising lipid A and a core oligosaccharide that includes an outer core, inner core, and a polysaccharide repeat (O antigen) [6,7]. A. baumannii possesses lipooligosaccharide (LOS), a molecule similar to LPS, but without the O antigen [8]. LPS/LOS appear as micelles or actively secreted membrane vesicles (MVs) when released into physiological fluids [7,9]. LPS/LOS monomers are extracted from MVs by LPS/LOS binding protein (LBP) and transferred to CD14 [10,11], the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex, followed by dimerization of the TLR4/MD-2 complex and activation of the MyD88 and/or TRIF signaling pathway [10,12].

Researchers believe that TLR4 signaling in LPS/LOS depends on the lipid A moiety [13,14] and that the core region does not significantly modulate this signaling [10]. Deletion of the lpsB gene results in loss of the LOS core region, leaving only 3-deoxy-D-manno-oct-2-ulsonic acid 9 (KDO) and lipid A on LOS (designated as Re-LOS hereafter) [7]. Compared with LOS, Re-LOS results in lower serum resistance in vitro, survival fitness in vivo [15], and virulence in a mouse pneumonia model [16].

In this study, we demonstrated that the core region of A. baumannii LOS contributes substantially to immune modulation by selectively enhancing one of the TLR4 signaling pathways. Mouse pneumonia and an intraperitoneal sepsis model with C57BL/6 mice were used in animal experiments to elucidate the pathogenesis of A. baumannii LOS. The LOS core region significantly promoted CD14-associated TLR4 endocytosis and consequently upregulated the expression of IFN-β, which correlated with increased pathogenicity in the animal model.

Results

Bacterial strains

The plasmid used for generating lpsB gene knockout A. baumannii mutants is shown in S1a Fig. S1b Fig shows a schematic representation of A. baumannii strains harboring different LOS lengths. S1c Fig presented PCR mapping results confirming the successful double-crossover deletion of the lpsB gene in strain Ab908ΔlpsB. Silver stain of the purified LOS confirmed that Ab908 and Ab908ΔlpsB::lpsB produce LOS with core, but not Ab908ΔlpsB (S1d Fig). The three strains exhibited comparable growth in vitro (S1e Fig).

0.1 A. baumannii with intact LOS demonstrated higher pathogenicity than those lacking the core region

A schematic diagram of the experimental design is shown in Fig 1a. Mice infected with strains harboring intact LOS displayed higher mortality rates (Fig 1b), lower body temperatures (Fig 1c), and greater lung weights (Fig 1d) than those that did not.

The bacterial load of the strain lacking the core region (Re-LOS) did not significantly differ from that of the strain with intact LOS at 4 h post-infection (Fig 1e). Serum white blood cell and neutrophil counts were significantly higher in mice exposed to the strain harboring an intact LOS 4 h post-infection (Fig 1f). The results showed that factors other than the bacterial burden may be responsible for the changes during the early phase of infection (4 h post-infection). In the serum and bronchoalveolar lavage fluid (BALF), the A. baumannii strain with intact LOS induced higher interferon beta (IFN)-β but not tumor necrosis factor (TNF)-α secretion (Fig 1g and 1h). Other cytokines/chemokines did not show consistent results in the serum and BALF (Fig 1g and 1h).

We used MVs in subsequent experiments to mitigate potential confounding factors associated with live bacteria that could influence pathogenicity (Fig 1i). The MVs released from the strains harboring intact LOS resulted in significantly higher fatality rates than those released from the strains with Re-LOS (Fig 1j). These results revealed that the LOS core region was associated with the enhanced pathogenicity of A. baumannii strains.

MVs containing intact LOS induced higher levels of IFN-β release and greater responsiveness of IFN-β-related genes in macrophages than those containing Re-LOS

We found that two cytokines, IFN-β and TNF-α, which are induced upon recognition of LOS by TLR4 [7,12], exhibited distinct responses in mice stimulated with A. baumannii harboring intact LOS versus Re-LOS. Therefore, we aimed to delineate whether strains carrying intact LOS or Re-LOS could elicit differential levels of IFN-β and TNF-α in immune cells. Macrophages were selected based on their important role as frontline innate immune cells during bacterial invasion and exposure to virulence factors.

The mouse macrophages stimulated with the MVs containing intact LOS secreted significantly more IFN-β than those stimulated with MVs containing Re-LOS, in a dose-dependent manner (Fig 2a). Moreover, the MVs with intact LOS elicited higher levels of Infb1 expression than those with Re-LOS did, in a dose-dependent manner (Fig 2b). The enhanced induction of IFN-β was consistent across the different A. baumannii strains belonging to various sequence types (Fig 2c). We showed that MVs containing intact LOS also induced significantly more IFN-β, IL-6, and IL-10 production in human macrophages (Fig 2d). In contrast, there was no significant difference in the production of TNF-α, IL-1β, and MIP-2 production among the groups (Fig 2d).

Fig 2 — **(a)** Interferon (IFN)-β secretion and **(b)** *Infb1* gene expression in murine macrophage J774a.1 cells stimulated with membrane vesicles (MVs) at the indicated concentrations for 4 **h. (c)** IFN-β secretion 6 h after stimulation with MVs (multiplicity of infection [MOI]=500) from *lps*B knockout (Δ*lpsB*) strains and their parent wild-type (WT) *A. baumannii* strains belonging to different sequence types. **(d)** Cytokine levels in THP1-Dual-derived macrophages 24 h after stimulation with MVs from different strains (MOI = 500). The means and standard error of the means from three biological replicates are shown, with significance being determined using an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. **(e)** Top 5 biological processes identified using gene set enrichment analysis comparing responses to MVs from Ab908 (with intact LOS) and Ab908ΔlpsB (with Re-LOS) **(f)** Heatmaps illustrating the expression of genes related to “response to IFN-β”, “cellular response to IFN-β”, and “negative regulation of type I interferon-mediated signaling pathway” in J774a.1 cell in response to the indicated stimuli. The scale numbers of the color keys represent log₂(FC). All data were retrieved from three biological replicates.

To examine the gene expressions, murine macrophage J774a.1 cells stimulated with MVs of Ab908 and Ab908ΔlpsB were used for RNA sequencing. RNA sequencing analysis identified 134 differentially expressed genes (DEGs) in the Ab908 MV group compared with the Ab908ΔlpsB MV group (with Re-LOS), of which 132 were upregulated and 2 were downregulated (S2a Fig). Infb1 expression was significantly upregulated in the Ab908 MV group (with an intact LOS; S2b Fig). Gene Set Enrichment Analysis (GSEA) revealed that the top five upregulated biological processes were “response to IFN-β,” “cellular response to IFN-β,” “negative regulation of type I interferon-mediated signaling pathway,” “adhesion of symbiont to host,” and “regulation of response to IFN-γ” as shown in Fig 2e. Heatmap analysis (Fig 2f) depicted that the expression of Irf1 and Infb1 was significantly upregulated in “response to IFN-β” and “cellular response to IFN-β,” whereas that of Stat2 was upregulated in “negative regulation of type I interferon-mediated signaling pathway.” These results showed that the MVs containing intact LOS induced higher levels of IFN-β release and greater responsiveness of IFN-β-related genes in macrophages than those containing Re-LOS.

Core region of LOS enhanced IFN-β production through the TLR4-TRIF signaling pathway

LPS located within complex particles, such as MVs and intact gram-negative bacteria, may selectively promote TRIF-dependent TLR4 signaling [17,18]. lpsB deletion results in the loss of the LOS core, potentially altering the structure of the bacterial membrane [19] and indirectly affecting the characteristics and content of the MVs released from the strain with Re-LOS. For example, the MVs of the strain carrying Re-LOS were slightly larger than those of the strain carrying intact LOS (S1 Table). These changes could be responsible for the lower production of IFN-β in the macrophages stimulated with the MVs containing Re-LOS. However, compared with the MVs containing Re-LOS, those with intact LOS still induced higher IFN-β levels even after chemical or physical disruption (Fig 3a). We further clarified that neither DNase I, RNase A, nor proteinase K treatment altered the differences in IFN-β production (Fig 3b). IFN-β was abolished by polymyxin B, indicating that LOS and Re-LOS of the MVs substantially influenced IFN-β secretion (Fig 3b). Addition of the TLR4 receptor inhibitor TAK-242 and MD-2 inhibitor L48H37 abolished MV-induced IFN-β secretion, suggesting that the IFN-β secretion induced by the MVs containing LOS and Re-LOS was associated with TLR4 and MD-2 (Fig 3c). While CD14 is required for LPS-induced TLR4 endocytosis, treatment with an anti-CD14 antibody abolished MV-induced IFN-β secretion (Fig 3d) but not MV-induced TNF-α secretion (Fig 3e). These results indicated that the LOS core region, but not the configuration or other components (DNA, RNA, proteins) of MVs, was responsible for increasing IFN-β secretion through the TLR4-MD-2 signaling pathway.

Fig 3 — **(a)** Interferon (IFN)-β secretion in J774a.1 macrophages pre-treated with Triton X-100, subjected to freeze–thaw cycles, or sonicated (2 cycles of 25 s each with 5 s pause at 30% power on ice) before being stimulated with Ab908 membrane vesicles (MVs). **(b)** IFN-β secretion in J774a.1 macrophages pre-treated with DNase I, RNase A, proteinase K, or polymyxin B for 30 min before being stimulated with MVs. **(c)** IFN-β secretion in THP1-Dual-derived macrophages pre-treated with TAK242 (Toll-like receptor 4 [TLR4] inhibitor) for 1 h and L48H37 (Myeloid differentiation factor-2 [MD-2] inhibitor) for 0.5 h before being stimulated with MVs. **(d)** IFN-β and **(e)** TNF-α secretion in THP1-Dual-derived macrophages pre-treated with anti-human CD14 antibody (hCD14) or anti-human IgG (hIgG, control) for 1 h before being stimulated with MVs (multiplicity of infection [MOI] = 500) from different bacterial strains. Cytokines were measured using an enzyme-linked immunosorbent assay 4 h after stimulation. The concentrations used were as follows: Triton X-100 (0.1% vol/vol); DNase I, 1 U/μL; RNase A, 200 μg/mL; proteinase K, 100 μg/mL; polymyxin B, 3 μM; TAK242, 1 μM; L48H37, 10 μM; hIgG, 10 μg/mL; hCD14, 10 μg/mL. Statistical significance was determined using an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01, ***P < 0.001.

LOS core region selectively enhances the TRIF-dependent pathway, but without affecting the MyD88-dependent pathway

TNF-α secretion was abolished by pre-treatment with polymyxin B, indicating that the MV-induced TNF-α production was also associated with LOS (Fig 4a). The MVs with intact LOS from isolates belonging to different sequence types stimulated equivalent TNF-α secretion (Fig 4b) compared with their Re-LOS mutants. These results suggest that the LOS core selectively enhances the TRIF-dependent signaling pathway but not the MyD88-dependent signaling pathway.

MVs containing either LOS or Re-LOS equally activated the NF-κB pathway (Fig 4c); however, only MVs with LOS enhanced the activation of the interferon regulatory factor 3 (IRF3) pathway (Fig 4d). Stimulation with LOS, but not Re-LOS, resulted in the phosphorylation of IRF3 at an early time point (Fig 4e). IFN-β production was eliminated after MRT67307 addition, an IKKε/TBK1 inhibitor (Fig 4f). These findings revealed that the MVs stimulated the release of IFN-β through the IRF3 pathway.

Several TRIF-dependent cytokine genes, including CXCL9, CXCL10, CXCL11, and Rsad2, were significantly upregulated after stimulation with MVs containing intact LOS (Fig 4g). MVs containing either LOS or Re-LOS induced similar levels of MyD88-associated gene expression (Fig 4h). IFN-β production was eliminated in a cell line lacking TRIF (Fig 4i) but preserved in the ones lacking MyD88 (Fig 4j), indicating that the LOS-enhanced IFN-β production was solely dependent on the TLR4-TRIF signaling pathway.

The top five enriched transcription factor (TF)-binding motifs were Stat2, Irf2, Irf1, Prdm1, and Irf3 (S2 Table), indicating that the upregulated DEGs were regulated by these TFs and may not be associated with the MyD88-dependent signaling pathway. Collectively, we demonstrated that the LOS core region selectively enhanced the TRIF-dependent signaling pathway but not the MyD88-dependent signaling pathway.

Core of LOS enhanced CD14-dependent TLR4 endocytosis

Dynasore (endocytosis inhibitor) obliterated IFN-β production, indicating that TLR4 endocytosis is necessary for MV-stimulated IFN-β secretion (Fig 5a). The inhibition of spleen tyrosine kinase (Syk) with piceatannol and its downstream phospholipase Cγ2 (PLCγ2) with U-73122 also eliminated the induction of IFN-β secretion by both MV stimulation groups (Fig 5a). These results indicate that LOS and Re-LOS induced IFN-β through CD14-dependent TLR4 endocytosis and TRIF-dependent signaling pathway. Unexpectedly, MVs with intact LOS enhanced the endocytosis of CD14 (Fig 5b) and TLR4 (Fig 5c), as well as TLR4/MD-2 dimerization (Fig 5d), compared with MVs with Re-LOS. These results revealed that LOS increased IFN-β production by promoting CD14-associated TLR4 endocytosis.

Fig 5 — **(a)** Interferon (IFN)-β measurement in THP1-Dual-derived macrophages (5 × 10⁵/well) pre-treated with dynasore (endocytosis inhibitor, 80 μM) for 1 h, piceatannol (Syk inhibitor, 75 μM), or U-73122 (PLCγ2 inhibitor, 5 μM) for 0.5 h before being stimulated with membrane vesicles (MVs) from different strains (multiplicity of infection [MOI] = 500, 4 h). n = 3 biological replicates per group. **(b)-(d)** Flow cytometry results of endocytosis of (b) CD14, (c) Toll-like receptor 4 (TLR4), and **(d)** TLR4/Myeloid differentiation factor-2 (MD-2) dimerization in J774a.1 cells stimulated with MVs for the indicated times. Error bars represent mean ± standard error of the mean (SEM) from triplicate experiments. MFI, mean fluorescence intensity. The means and standard error of mean from three biological replicates are presented, with significance determined by unpaired two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001. LPS, lipopolysaccharide.

IFN-β stimulated by the MVs with intact LOS is associated with increased pathogenicity

Wild-type C57BL/6 and IFNAR1-deficient mice (Ifnar1-/-), which lacked the type I IFN-α/β receptor, were intraperitoneally injected with MVs containing either intact LOS or Re-LOS. The results showed that IFNAR1-deficient mice had a higher survival rate than wild-type mice when challenged with MVs containing intact LOS (Fig 6a). However, the survival of IFNAR1-deficient mice differed significantly between the mice challenged with MVs containing intact LOS and those challenged with Re-LOS-containing MVs. Treatment with an anti-IFN-β antibody significantly reduced mortality in mice exposed to MVs with intact LOS, in a dose-dependent manner, compared with mice treated with an isotype control antibody (Fig 6b). These findings indicate that LOS-induced IFN-β production contributes to the increased pathogenicity of A. baumannii in mice. In addition to IFN-β, intact LOS may also contribute to increased pathogenicity through additional mechanisms.

Fig 6 — **(a)** Survival rates of female C57BL/6 and IFNAR1-deficient mice (Ifnar1-/-) that were intraperitoneally injected with *A. baumannii* MVs containing intact LOS/Re-LOS (Ab908/Ab908ΔlpsB) (~9.7 × 10¹¹ particles in 100μL; n = 10–12 in each group). **(b)** Survival rates of female C57BL/6 mice that were intraperitoneally injected *A. baumannii* with intact LOS (Ab908) MVs (~9 × 10¹¹ particles in 100μL). Anti-interferon-β antibody and isotype antibody were used 30 min after MVs injection (n = 16 in each group). Kaplan–Meier analysis was conducted. (*P < 0.05, log–rank test).

Discussion

Gram-negative bacteria such as Neisseria meningitidis, Haemophilus influenzae, and A. baumannii produce LOS, which play a critical role in evading host immune responses. The core region of LOS often mimics the host cellular components, enabling these bacteria to avoid detection [7,16]. We found an extended role for the LOS core in immune modulation, particularly in its ability to selectively enhance one of the TLR4 signaling pathways. Specifically, the core region of A. baumannii LOS significantly promoted CD14-associated TLR4 endocytosis and augmented IFN-β production via the TRIF-dependent signaling pathway, ultimately contributing to increased pathogenicity (Fig 7).

Fig 7 — Schematic illustration of the findings in this research. Created with BioArt Source (https://bioart.niaid.nih.gov/) and Wikimedia Commons (https://commons.wikimedia.org/).

Our results demonstrated that certain cytokines/chemokines showed inconsistent levels in mouse serum and BALF. These discrepancies may reflect differences in the extent of local airway versus systemic inflammation [20], as well as inter-individual variability among mice. Several studies have reported that cytokine levels in BALF and serum do not always increase consistently in patients with pneumonia [21,22]. However, the two cytokines IFN-β and TNF-α, which are induced upon recognition of LOS by TLR4, exhibited distinct responses both in blood and BALF, prompting us to further investigate this phenomenon using a cell model.

Lipid A is traditionally considered the primary moiety of LPS/LOS that modulates TLR4 signaling because of its acyl and phosphate groups, which directly interact with MD-2 and TLR4 [10]. Modifications of lipid A can significantly affect TLR4 signaling [23]. For example, hypoacylated LPS reduces the activation of MyD88- and TRIF-dependent signaling pathways [24]. Recently, findings showed that the KDO moieties of the inner core interact with TLR4, enhancing the potency of lipid A in TLR4 activation [25–27].

However, the role of the saccharide components of LPS/LOS in TLR4 signaling modulation remains poorly understood. For example, the O antigen of Salmonella LPS delays TLR4-mediated recognition and enhances the resistance to macrophage phagocytosis [28,29]. Our results indicate that because of the LOS core, TLR4 signaling can be specifically biased toward the TRIF-dependent signaling pathway without affecting the MyD88-dependent signaling pathway. A previous study revealed that the intact LPS of Escherichia coli (smooth LPS [sLPS]) and its Re-LPS mutant were similarly efficient in activating the MyD88-dependent pathway. However, sLPS was slightly more efficient than Re-LPS in activating the TRIF-dependent pathway [30]. Additionally, LPS core truncation in E. coli has been associated with a reduced ability to invade epithelial cells [30]. Pseudomonas aeruginosa loses its core saccharides during chronic infection [31]. These findings reveal that the core region of LPS/LOS can modulate specific signaling pathways that contribute to pathogenicity.

Our results show that TLR4 could not differentiate between LOS and Re-LOS cells when CD14 expression was blocked. Thus, CD14 can discriminate between LOS and Re-LOS. Similarly, CD14 has been shown to distinguish between LPS with different saccharide structures [32], demonstrating that LPS with the O antigen (sLPS) is more dependent on CD14 than that without the O antigen (rLPS) for transport to TLR4, thereby initiating the MyD88-dependent signaling pathway [30,33]. Additionally, sLPS is more efficient in activating CD14-endocytosis-associated nuclear factor of activated T-cell pathways in dendritic cells [30]. These findings indicate that CD14 recognizes specific saccharide components of LPS/LOS to exert distinct immunomodulatory functions.

While IFN-β is beneficial in most acute viral infections, its role in bacterial infections can be either advantageous or harmful, depending on factors such as bacterial species, burden, duration, and location of induction [34,35]. Our results showed that enhanced IFN-β production by the core region of A. baumannii LOS correlated with increased pathogenicity. Using mice stimulated with E. coli MVs, it was previously demonstrated that genetic deletion of TRIF reduced lethality, indicating that the TRIF-dependent signaling pathway is linked to higher mortality [36].

Not all IFNAR1-deficient mice (Ifnar1-/-) survived the challenge with MVs containing intact LOS, and their survival rates were lower than those of mice challenged with MVs containing Re-LOS. These findings reveal that, in addition to IFN-β, intact LOS may contribute to increased pathogenicity through additional mechanisms. For example, an intact LOS may enhance resistance to phagocytosis or complement-mediated disruption [7,16]. Further studies using IFNAR1-deficient cells may help elucidate additional mechanisms by which the LOS core contributes to increased pathogenicity.

Studies have explored the potential development of novel drugs and vaccines targeting the LPS-TLR4 signaling pathway. TLR4 activators are potential therapeutic immunomodulators and adjuvants [37]. LPS neutralization and TLR4 blockade are also promising therapeutic strategies [38]. We showed, in this study, that using the LOS core region as a vaccine candidate or targeting the LOS core, its synthetic enzymes, and the interface between the LOS core and CD14 to develop drugs or antibodies may potentially aid in combating A. baumannii.

This study has some limitations. While enhanced CD14-associated TLR4 endocytosis increases IFN-β production [39,40], the mechanism by which LOS enhances CD14 endocytosis remains unclear. Other receptors, such as lectins, may recognize the saccharide components of LPS and contribute to CD14-associated TLR4 endocytosis. Moreover, the molecular structural differences between LOS and Re-LOS and their interactions with CD14 were lacking in this study. Further structural biology techniques, such as X-ray crystallography, molecular docking, nuclear magnetic resonance spectroscopy, and mass spectrometry, should be considered to provide a more detailed structural characterization of the interactions between LOS and CD14.

Conclusion

Our study highlights the pivotal role of the core region of A. baumannii LOS in enhancing TLR4 endocytosis, leading to increased IFN-β production and virulence. These findings could influence the development of novel therapeutic strategies targeting the LOS core to effectively combat A. baumannii infections.

Materials and methods

Ethics statement

The protocols of the study were approved by the Laboratory Animal Center of the National Defense Medical University (IACUC-21–33 and IACUC-22–236).

Bacterial strains

Bacterial strains used in this study are listed in S3 Table. The multi-locus sequence typing scheme involving polymerase chain reaction (PCR) amplification and sequencing of seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) was performed on the selected isolates as previously described [41]. Strains with Re-LOS were constructed as previously described [42,43]. The lpsB gene in A. baumannii genomic DNA was disrupted by inserting an apramycin antibiotic resistance gene (Apr^R) to generate knockout mutants. Briefly, the Apr^R integration cassette was obtained with PCR amplification from the pGEM-T-Easy Vector DNA template using P1 and P2 primers. Approximately 500 bp regions in R1 and R2, corresponding to the 5′-proximal and 3′-proximal regions of the lpsB coding sequence, were amplified using P3/P4 (R1 region) and P5/P6 (R2 region) primers. The three PCR products were subsequently combined (S1a Fig) and reamplified to generate a linear fragment containing an apramycin resistance cassette flanked by two specific regions. The linear product was cloned into a pGEM-T Easy Vector. The plasmid was taken up into A. baumannii competent cells by electroporation and subsequently selected on Luria-Bertani (LB) agar plates supplemented with 50 μg/mL apramycin. The successful generation of these ΔlpsB mutants carrying Re-LOS was verified using PCR (S1c Fig). The primer sequences are listed in S4 Table. To complement the mutant phenotype, the wild-type lpsB gene was cloned into an A. baumannii-E. coli shuttle vector pAbYm2 [44] to generate pAbYm2lpsB. The shuttle vector pYMAb2 was previously generated in our lab and derived from E. coli plasmid pET-28a, which has a kanamycin resistant determinant and an SphI/XbaI fragment containing replicons from A. baumannii ATCC19606 plasmid pMAC [44]. The vector pAbYm2lpsB was electroporated into the ΔlpsB mutant to construct the complement strain, ΔlpsB::lpsB. The successful generation of these mutants was verified using PCR.

To better display the presence and absence of the LOS in the bacterial strains. Two E. coli keio knockout strains were purchased from Horizon discovery (Cambridge, UK) and used for the silver staining of LOS. E. coli BW25113 lacking waaL gene (EcΔrfaL) produces lipopolysaccharides deficient in the O-antigen was used as a reference of Ab908. In addition, E. coli BW25113 lacking waaC gene (EcΔrfaC) produces truncated lipopolysaccharides, specifically losing the outer core and parts of the inner core was used as a reference of Ab908ΔlpsB [45].

Silver staining of LOS

LOS from A. baumannii and E. coli were extracted using an LPS extraction kit (Boca Scientific Inc., Waltham, MA, USA). The extracted samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) and RNase A (25 μg/mL; QIAGEN, Hilden, Germany) at 37 °C for 2 h to remove contaminating nucleic acids, followed by incubation with proteinase K (100 μg/mL; Sigma–Aldrich, St. Louis, MO, USA) at 37 °C overnight to eliminate residual proteins. The samples were then mixed with 4 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and heated at 95 °C for 5 min prior to electrophoresis. Purified LOS were separated on 20% sodium dodecyl sulfate–polyacrylamide gels (SDS-PAGE) and visualized using a Pierce Silver Stain kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Briefly, gels were washed twice with ultrapure water (5 min each), fixed twice in 30% ethanol and 10% acetic acid (15 min each), and then oxidized in a solution containing 1% periodic acid in 30% ethanol and 10% acetic acid for 20 min. The gels were subsequently washed twice in 10% ethanol (5 min each), followed by three washes in ultrapure water (5 min each). For sensitization, gels were treated with sensitizer solution for 1 min and washed twice. Gels were then stained with a mixture of enhancer and stain solution for 30 min and developed using enhancer and developer solution for 5 min. The reaction was terminated by incubation in 5% acetic acid for 10 min.

Growth of the isolates

Briefly, 10 mL of LB broth was inoculated with a 100 μL overnight culture and incubated at 37 °C with shaking at 200 revolutions per min (rpm) for 6 h. Aliquots were obtained at 0, 1, 2, 4, 6, and 24 h post-inoculation, serially diluted, and plated on trypticase soy agar (TSA) plates to determine the viable cell counts.

Cell lines and culture conditions

Human THP1-Dual, THP1-Dual KO‑TRIF, and THP1-Dual KO-MyD88 were purchased from InvivoGen (San Diego, CA, USA). J774a.1 murine macrophage cells were purchased from Sigma–Aldrich (St. Louis, MO, USA). The human THP1-Dual, THP1-Dual KO‑TRIF, and THP1-Dual KO-MyD88 cells were derived from the Human THP1 monocyte cell line by stable integration of an IRF-luciferase reporter and NF-κB alkaline phosphatase (secreted embryonic alkaline phosphatase, SEAP) reporter. The cells were used for simultaneous study of the NF-κB pathway, by monitoring the activity of SEAP, and the IRF pathway, through assessment of the activity of Lucia luciferase.

All the cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 100 μg/mL Normocin, and Penicillin-Streptomycin solution (100 U/mL-100 μg/mL). Cultures were incubated in a humidified incubator at 37 °C using 5% carbon dioxide.

To differentiate the macrophage phenotype, THP1-Dual, THP1-Dual KO-TRIF, and THP1-Dual KO-MyD88 cells were treated with phorbol 12-myristate 13-acetate (PMA) at a concentration of 50 ng/mL in cell culture plates (approximately 5 × 10⁶ cells/mL) for 24 h [46]. Following incubation, PMA was removed.

Chemical sources

Triton X-100, polymyxin B, L48H37 (MD-2 inhibitor), and U-73122 (PLCγ2 inhibitor) were purchased from Sigma–Aldrich (St. Louis, MO, USA). DNase I and RNase A were purchased from QIAGEN (Hilden, Germany). TAK242 (TLR4 inhibitor) was purchased from Merck (Darmstadt, Germany). Dynasore (endocytosis inhibitor) was purchased from Abcam (Cambridge, UK). Piceatannol (a Syk inhibitor) was purchased from Bio-Techne (Minneapolis, MN, USA). MRT67307 (IKKε/TBK1 inhibitor), hIgG (anti-human IgG), and hCD14 (purified anti-human CD14 antibody) were purchased from InvivoGen (San Diego, CA, USA). The anti-IFN-β antibody and mouse IgG2a (C1.18.4) isotype control antibody for animal experiments were purchased from Ichorbio (Wantage, UK). The Cell Counting Kit (CCK kit) was purchased from DOJINDO Laboratories (Kumamoto, Japan).

Animal experiments

Wild-type C57BL/6 mice were purchased from the National Laboratory Animal Center (NLAC, Taipei, Taiwan). The IFNAR1-deficient mice (Ifnar1-/-) were kindly provided by Dr. Guann-Yi Yu (National Health Research Institutes, Taiwan). All the mouse experiments were conducted following the recommendations of the Laboratory Animal Center of the National Defense Medical University. The ARRIVE guidelines were adhered to for all mouse experiments, and the protocols were approved by the Laboratory Animal Center of the National Defense Medical University (IACUC-21–33 and IACUC-22–236). The experiments were carefully designed to minimize the number of animals used while ensuring statistical significance. Appropriate anesthesia and analgesia were administered to alleviate potential discomfort during the procedure.

Isolation and purification of MVs

MVs were isolated from late log phase cultures (16 h) of A. baumannii. Briefly, 400 mL of LB broth was inoculated with 4 mL of an overnight culture and incubated at 37 °C with shaking at 200 rpm for 6 h. The cells were pelleted by centrifuging at 12,250 × g for 20 min, and the supernatant was filtered through a 0.22 μm membrane filter (JET BIOFIL, China) to remove cells and cellular debris. The filtrate was subjected to ultracentrifugation at 142,000 × g for 2 h at 4 °C using a Type 45 Ti rotor (Beckman, USA). To wash the MVs, the pellet was resuspended in Phosphate-buffered saline (PBS) and ultracentrifuged again at 142,000 × g for 2 h at 4 °C using the same rotor. The final pellet was resuspended in 800 μL of PBS and kept on ice. The resulting MV suspension was cultured on Mueller-Hinton agar and confirmed to be free of bacteria.

Characterization of MVs using a nanoparticle tracking analyzer

The nanoparticle tracking analyzer (NTA) device, ZetaView PMX120 (Particle Metrix GmbH, Meerbusch, Germany), was used to measure the size distribution of MVs at 25 °C. Polystyrene standard beads (Thermo Fisher Scientific, Waltham, MA, USA) were used to align the focus and camera/laser positions. Samples were diluted in PBS to 1 mL and measured at 25 °C. Each sample was scanned at 11 cell positions, and particle sizes were calculated based on their Brownian motion. Data were processed using ZetaView software v8.05.10. Measurements were performed in scatter mode using a 488 nm laser, while fluorescence was detected in fluorescence mode using the same laser with a 500 nm long-pass filter. All experiments were performed in triplicate.

Mice pneumonia model

Seven-week-old female C57BL/6 mice were injected intratracheally with strains of A. baumannii. The bacterial inoculum (colony-forming units, CFU) was determined from aliquots of suspended bacteria that were serially diluted and plated onto TSA for enumeration. In the first experiment, mice were monitored every 12 h for 72 h after bacterial exposure for signs of morbidity. The mice were sacrificed, and the lungs were harvested and weighed. In the second experiment, mice were euthanized 4 h after bacterial exposure. The lungs were harvested, homogenized, and plated on TSA to enumerate bacterial counts. Blood samples were collected and subjected to differential white blood cell counts. The serum and BALF of mice were obtained 4 h after bacterial exposure and sent for cytokine and chemokine determination in the third experiment.

Mice intraperitoneal sepsis model

Three experiments were performed using a mouse model of intraperitoneal sepsis. In the first experiment, seven-week-old female C57BL/6 mice were instilled intraperitoneally with MVs (~8.6 × 10¹¹ particles in 100 μL) from different A. baumannii strains. Mice were monitored for signs of morbidity every 12 h for 96 h after bacterial exposure. In the second experiment, 7-week-old female C57BL/6 and IFNAR1-deficient mice (Ifnar1-/-) were instilled intraperitoneally with MVs (~9.7 × 10¹¹ particles in 100 μL) from Ab908 and Ab908ΔlpsB. The mice were monitored for signs of morbidity every 12 h for 96 h after MVs instillation. In the third experiment, 7-week-old female C57BL/6 mice were instilled intraperitoneally with MVs (~9 × 10¹¹ particles in 100 μL) from Ab908. The anti-IFN-β antibody (300 μg or 600 μg) or IgG2a (C1.18.4) isotype control antibody (600 μg) was instilled intraperitoneally 30 min later. Mice were observed for signs of morbidity every 12 h for 96 h.

Enzyme-linked immunosorbent assay for cytokines

THP1-Dual or J774a.1 cells were seeded in a 24-well plate at a density of 5 × 10⁵ cells/well and cultured overnight with 50 ng/mL PMA. The following day, cells were washed with PBS and cultured in fresh RPMI 1640 medium overnight. For inhibition experiments, cells were pre-treated with the following inhibitors at the specified concentrations and durations: DNase I (1 U/μL), RNase A (200 μg/mL), polymyxin B (3 μM), K (100 μg/mL), L48H37 (10 μM), piceatannol (75 μM), and U73122 (5 μM) for 0.5 h; TAK242 (1 μM), hCD14 (10 μg/mL), hIgG (10 μg/mL), dynasore (80 μM), and MRT67307 (10 μM) for 1 h. After pre-treatment, the cells were stimulated with MVs. Subsequently, the supernatants were collected 4 h after stimulation, and cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Diagnostics, Minneapolis, MN, USA).

RNA extraction and quantitative real-time polymerase chain reaction

Quantitative Real-time polymerase chain reaction was used to determine cytokine gene expression levels. Briefly, macrophages were stimulated with MVs for 4 h, and total RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen, Netherlands) following the manufacturer’s instructions. cDNA was generated by the reverse transcription of 1–2 μg of total RNA using oligo dT primers and M-MuLV reverse transcriptase in a total reaction volume of 20 μL (Thermo Fisher Scientific, Waltham, MA, USA). Gene transcripts were quantified using the Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Amplification specificity was evaluated using melting curve analysis. The primer sequences are listed in S4 Table.

RNA sequencing and data analysis

J774a.1 cells were seeded in plates and subsequently stimulated with MVs of Ab908 and Ab908ΔlpsB (MOI = 500) for 2 h. The cells were harvested at the end of stimulation, which was followed by RNA extraction. The purity and quantity of RNA were checked using a SimpliNanoBiochrom spectrophotometer (Biochrom, MA, USA). RNA degradation and integrity were monitored using a Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). One microgram of total RNA per sample was used as input material for the RNA sequencing sample preparations. Sequencing libraries were generated using the KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland), following the manufacturer’s recommendations. The index codes were added for the unique identification of each sample. The sequences were determined using the Illumina NovaSeq 6000 platform. Sequencing data (FASTQ reads) were generated using bcl2fastq v2.20, followed by adaptor clipping and sequence quality trimming using Trimmomatic (v0.36) [47]. Filtered reads were mapped to mm10 using HISAT2 software [48]. Genes in the mapped reads were identified using featureCounts [49]. DEGs, GSEA biological processes, and TF-binding motifs were analyzed using integrated Differential Expression and Pathway analysis [50].

Biological activity

QUANTI-Luc (rep-qlc1) was used to detect the level of luciferase by adding it to the culture supernatant and reading immediately using a plate reader (SpectraMax iD5; Molecular Devices, California, CA, USA) at a 0.15-s reading time. QUANTI-Blue (rep-qb1) was used to detect the level of SEAP by adding it to the culture supernatant, incubating for 30 min, and measuring the absorbance with a plate reader (SpectraMax iD5) at 620 nm. Quanti Luc and Quanti Blue were purchased from InvivoGen (San Diego, CA, USA)

Cytotoxicity of the inhibitors

The CCK kit was used to evaluate the cytotoxicity of the inhibitors following the manufacturer’s recommendations. Briefly, J774a.1 cells were grown in 96-well plates at 12,500 cells/well in Dulbecco’s Modified Eagle Medium (DMEM). Overnight cell cultures were washed and replaced with fresh DMEM. The cells were pretreated with the indicated concentrations of the inhibitors for 1 h. Wells containing DMEM alone were used as controls. MVs of the three isolates were added to 96-well plates. Following a 6-h exposure to the MVs, CCK solution was added to each well, and the plates were re-incubated for 1 h. Finally, 10 μL 0.1 M HCl was added to each well to stop the reaction. The number of viable cells was assessed by measuring absorbance at 450 nm using a microplate spectrophotometer.

Western blot analysis

J774a.1 cells were seeded in 6-well (1 × 10⁷ cells/mL) plates, followed by adding 1 µg/mL LPS and MVs. After incubation for 1 h or 6h, the total proteins were extracted using a protein extraction kit [51]. Protein concentrations were measured using a Bradford Protein Assay kit. Following this, equal amounts of protein (50 µg) from each sample were heated to 95˚C for 5 min with 4 × Laemmli sample buffer and subsequently separated using SDS-PAGE (10% gel). Proteins were transferred onto polyvinylidene fluoride membranes. Following blocking for 1 h with protein-free blocking buffer at room temperature, the membranes were incubated with primary antibodies against GAPDH, IRF3, and phosphorylated IRF3 overnight at 4˚C. Following washing three times with Tris-Buffered Saline with Tween-20 buffer, the membranes were incubated with the aforementioned secondary antibodies for 1 h at room temperature. The electrochemiluminescence kit and GAPDH antibody were purchased from GeneTex International Corporation (Hsinchu, Taiwan). The phospho-IRF-3 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The IRF3 antibody was purchased from Abcam (Cambridge, UK).

Flow cytometry for TLR4 endocytosis and TLR4/MD-2 dimerization

TLR4 endocytosis and TLR4/MD-2 dimerization were measured using flow cytometry [24]. Briefly, J774a.1 cells were seeded in well plates and stimulated as previously indicated at 37 °C. The cells were subsequently washed and stained with the appropriate antibodies, and bovine serum albumin was used to reduce nonspecific binding of the antibodies. The surface receptors were stained using a BD FACSCanto II. The mean fluorescence intensities (MFIs) of CD14 and TLR4 in unstimulated and stimulated cells were recorded. The percentage of TLR4/MD-2 dimer formation was calculated as follows: 100%－the percentage of TLR4/MD-2 monomer formation; this percentage was determined as the ratio of the MFI values of stimulated cells to those of unstimulated cells.

Statistical analysis

For continuous variables, the Student’s t-test was used to assess differences. Time to mortality was analyzed using the Kaplan–Meier survival analysis and the log–rank test. All analyses were processed using SPSS version 26.0.

Supporting information

S1 Table. 1. The size of membrane vesicles from different strains used in this study.

(DOCX)

ppat.1014364.s001.docx^{(15.5KB, docx)}

S2 Table. 2. Enriched transcription factor (TF) binding motifs in promoters of differentially expressed genes (DEGs).

(DOCX)

ppat.1014364.s002.docx^{(18KB, docx)}

S3 Table. Bacterial isolates used in this study.

(DOCX)

ppat.1014364.s003.docx^{(18.2KB, docx)}

S4 Table. The primers used in this study.

(DOCX)

ppat.1014364.s004.docx^{(18.9KB, docx)}

S1 Fig. The structure of the plasmid, the PCR mapping for the confirmation of successful deletion of lpsB gene, the lipooliosaccharide (LOS) structure, and the growth of the bacterial strains used in this study.

Schematic presentation of the plasmid used for the generating the lpsB gene knockout Acinetobacter baumannii mutant (a). Schematic presentation of the A. baumannii strains harboring different length of LOS (b). PCR mapping for the confirmation of successful deletion of lpsB gene (c). Silver stain of the purified LOS shows that A. baumannii 908 (Ab908), the complementary strain (Ab908ΔlpsB::lpsB), and Escherichia coli BW25113 lacking waaL gene (EcΔrfaL) produce LOS with core, but not the lpsB deletion mutant strains (Ab908ΔlpsB) and EcΔrfaC (d). Growth curve of the three A. baumannii strains (e).

(TIF)

ppat.1014364.s005.tif^{(774.2KB, tif)}

S2 Fig. RNA sequencing analysis of the gene expression.

RNA sequencing analysis showed differential gene expression after stimulation with membrane vesicles (MVs) from the wild-type A. baumannii strain Ab908 (harboring intact LOS) and Ab908ΔlpsB (harboring Re-LOS) (a). Volcano plots depicting gene expression changes in J774a.1 cells, showing fold change and P value comparisons after stimulation with MVs from Ab908 and Ab908ΔlpsB (b).

(TIF)

ppat.1014364.s006.tif^{(358.1KB, tif)}

S3 Fig. Cytotoxicity of the inhibitors used in this study.

The J774a.1 cells were grown in 96-well plates at a density of 12,500 cells per well and treated with inhibitors with indicated concentrations for 1 h. The membrane vesicles (MVs) of three different isolates were then added to the 96-well plates. After 6 h exposure to the MVs, the number of viable cells was assessed by measurement of the absorbance at 450 nm using a microplate spectrophotometer. The concentration of the inhibitors used were as following: DNase I, 1 U/μL; RNAse A, 200 μg/mL; polymyxin B, 3 μM; TAK242, (TLR4 inhibitor, 1 μM); L48H37, (MD-2 inhibitor, 10 μM); MRT67307 (IKKε/TBK1 inhibitor, 10 μM), dynasore (endocytosis inhibitor, 80 μM), piceatannol (Syk inhibitor, 75 μM), U-73122 (PLCγ2 inhibitor, 5 μM); PBS, phosphate-buffered saline.

(TIF)

ppat.1014364.s007.tif^{(295.1KB, tif)}

Acknowledgments

We are grateful to Dr. Guann-Yi Yu for supplying the IFNAR1-deficient mice. We would also like to acknowledge the Laboratory Animal Center of National Defense Medical University (NDMU-LAC), Taiwan, for support in animal husbandry and medical care.

Data Availability

The data that support the findings of this study are openly available in figshare at doi:10.6084/m9.figshare.28028600.

Funding Statement

This work was supported by the Taipei Veterans General Hospital through grants VTA113-V1-2–1 and VTA114-V1-2–1 (to Y.T.L.); by the Tri-Service General Hospital through grants TSGH-E-113286, VTA113-V1-2–2, TSGH-E-114278, VTA114-V1-2–2, and TSGH-E-115280 (to Y.C.W.); by the Ministry of Science and Technology through grants MOST-110–2314-B-016–063 and MOST-111–2314-B-016–034 (to T.L.C.); and by the National Science and Technology Council through grants NSTC-112–2314-B-016–024-MY2 and NSTC-114–2314-B-016–030 (to T.L.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1014364.r001

Decision Letter 0

Matthew Wolfgang, Mario Feldman

25 May 2025

-->PPATHOGENS-D-25-00850

Acinetobacter baumannii lipooligosaccharide core region enhances CD14-dependent TLR4 endocytosis and boosts virulence through interferon-β production

PLOS Pathogens

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 60 days Jul 24 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

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If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Mario F Feldman

Academic Editor

PLOS Pathogens

Matthew Wolfgang

Section Editor

PLOS Pathogens-->--> -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

-->-->Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

Three experts in the topic have revised this manuscript. The three of them found the work interesting. However, they have raised concerns that need to be addressed in order to fully support the authors' conclusions. In particular, the work with OMV needs more quantifications/controls. Visualization by microscopy and proper LOS/protein quantifications are needed. The affinity of CD14 to the different LOS needs to be investigated by other methods. In Figure 4e, the GAPDH loading controls must be shown for the 1 h samples and the 6 h samples. For all experiments using inhibitors, controls showing maintenance of viability or secretion of other cytokines/chemokines are needed. E.g. Fig 3b, Fig 3c, Fig 4f, Fig 4i, Fig. 5a. Finally, there are concerns about the results in Figure 6 that require more work to be conclusive. Details pm experimental procedures including bacterial inoculum (CFU), reagents and inhibitors concentration, as well as complete procedures are missing from Material and Methods.

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Yi-Tzu Lee, Te-Li Chen, Shu-Chen Kuo, Yu-Han Liu, Meng-Chang Lee, Ming-Fen Chuang, Mei-Chei Tan, I-Ming Lee, Ya-Sung Yang, and Yung-Chih Wang. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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- TM on pages: 32, 33, 34, and 35.

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3) If any authors received a salary from any of your funders, please state which authors and which funders..

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In the manuscript, Acinetobacter baumannii lipooligosaccharide core region enhances CD14-dependent TLR4 endocytosis and boosts virulence through interferon-β production, Lee and Chen et al. demonstrate that the core region of A. baumannii LOS stimulates an immune response. This finding negates previous dogma that only Lipid A is the stimuli required for LPS/LOS derived inflammation. As such, I believe this work should be published in a high-tier journal such as PLoS Pathogens. I only request that the questions below be addressed:

Reviewer #2: In this work, Yi-Tzu Lee et al. investigate the role of the core region of A. baumannii LOS in host immune response. They use membrane vesicles from the different A. baumannii strains (WT, ∆lpsB and complemented strain) to show that the presence of a full core LOS binds better to CD14 and leads to CD14-dependent TLR4 endocytosis. Finally, the authors show that increased survival of mice challenged with A. baumannii membrane vesicles correlates with decreased levels of interferon-B production proposing a central role of LOS core in A. baumannii virulence.

Reviewer #3: This manuscript dissects the effect of the A. baumannii LOS core region on different types of TLR4 signaling. Specifically, using an lpsB mutant defective in producing the LOS core, data show that that MyD88-depedent signaling is unaffected while CD14 binding and CD14-dependent signaling is reduced, suggesting that the A. baumannii LOS core promotes IFN-B production and virulence. These data are overall strong and convincing.

It seems that the major finding is that in addition to CD14 being required for recognizing smooth LPS from e.g. E. coli, CD14 is also able to bind and differentiate between A. baumannii intact LOS (with a much shorter polysaccharide region) and re-LOS with only KDO on lipid A. A schematic figure may help clarify the major finding of the study.

In summary, this manuscript elucidates a role for CD14-dependent TLR4 discrimination between intact A. baumannii LOS with core saccharides and KDO-lipid A, further expanding our understanding of TLR4 signaling with implications for numerous gram-negative pathogens that encode LOS. However, the specific link between LOS core, CD14-dependent signaling, IFN-B production and virulence are not directly tested.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Lines 49-50 – Have the role of MVs for A. baumannii been shown in vivo?

Line 130-132 – While MVs have the known differences in core oligosaccharide content, it is unclear if differences in protein content could lead to a potential phenotype.

Lines 339-350 – It is unclear how MVs are quantified. Given the different amounts of LPS, there could be different amounts of MVs produced, skewing possible results. If MVs were not quantified microscopically, results could be questionable due to inconsistent dosage.

Line 650 – 40 ug of MVs is questionable when content of the MVs could greatly differ between WT and mutant bacteria, despite being relatively similar in size.

Reviewer #2: - Growth of the wildtype, ∆lpsB and complemented strains in the conditions of infections should be added to the supplementary as difference in LOS could lead to decrease fitness, impacting the interpretation of experiments in Figure 1.

- Although the pull down gives a qualitative result on the binding of CD14 to LOS, the authors should use an additional more quantitative method to determine the affinity of CD14 to the different LOS

- A lot of details on experimental procedures including bacterial inoculum (CFU), reagents and inhibitors concentration, as well as complete procedures are missing from the material and method, complicating the understanding of some procedures.

- The text should be edited to ensure language as well as scientific clarity.

Reviewer #3: 1. Experimental details with membrane vesicles are not clear, e.g. mouse experiment in Figure 1, Figure 2, Figure 3, .It is not clear how the MV were quantified and normalized for the mouse experiment. The inocula should be normalized for appropriate interpretation. Related, Table S1 shows the lpsB mutant has increased size of MV—how was this measured? If it was by microscopy, please display the images.

2. Some additional controls are needed: In Figure 4e, the GAPDH loading controls must be shown for the 1 h samples and the 6 h samples. For all experiments using inhibitors, controls showing maintenance of viability or secretion of other cytokines/chemokines are needed. E.g. Fig 3b, Fig 3c, Fig 4f, Fig 4i, Fig. 5a.

3. The link between LOS, IFN-B production, and virulence, remains inferred rather than directly interrogated. This should be addressed in the title and results. For Figure 6, the results section over concludes the linking of LOS core-dependent IFN-b signaling to A. baumannii virulence, “These results indicated that the LOS-enhanced IFN-β production was associated with the increased virulence of A. baumannii in the mice.” To test that, IFN-b and/or IFNAR-/- mice could be combined with A. baumannii lpsB mutants: if the model is correct, there should be no difference in virulence between MV from WT v. lpsB in IFNAR-/- or anti-IFN-b-treated mice. Additionally, it is not clear how the fact that IFNAR-/- also blocking IFN-a production would obscure a role for IFNAR in LOS- and CD14-dependent IFN-b production being important. Finally, for Figure 6b, the appropriate control is isotype antibody rather than untreated.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Fig. 1 – Bacteria in figures do not resemble A. baumannii (flagella should not be present).

Fig. 1C – Unclear if statistical testing is between WT vs. mutant, or Complement vs. Mutant.

Line 124 – It is unclear why bacterial burdens would be tested at 4 h when burdens would most likely be different later during infection

Lines 127-129 – Why are IL-1B, IL-6, IL-10, and KC ignored in the results section, when clearly something strange appears to be happening with these levels?

Supplementary Fig. S2b – Infb1 is not highlighted in the volcano plot.

Line 150-152 – This is not a complete sentence.

Fig. 2F – One of the WT heat maps looks completely different from the other 2 replicates. Can the authors explain this?

Lines 213-216 – The inhibitors used are not readily known by some readers, so more explanation may be necessary.

Line 320 – Can the sources, isolation site, isolation year, and the “many antimicrobials” resistant to be noted? This could be of great value to the field.

Lines 340-345. – Centrifugation speed should always be given in x g, as RPM varies by centrifuge.

Line 602 – Does MOI refer to 500 MVs/cell or 500 cells producing MVs/cell

Reviewer #2: - Following experiments in mice, the authors choose to investigate the response of macrophages, and they then alternate between experiments in THP-1 vs J774.A1 cells, it is unclear whether there is a rational for the use of one or the other cell type.

- Some figure sub-panels are not discussed in the text

- The authors do not discuss the inconsistency of other cytokines in between the different experiments. Could the authors speculate on these differences?

- Figure legends should include the description of abbreviations used in the figures

- Kinetics of killing using IP membrane vesicle appears rather inconsistent between experiments, how do the authors choose/normalize the number of vesicles injected?

Did the authors verify that the MV had a similar protein content? What quantity of MV is released by a certain number of bacteria?

- The authors should increase the size of the Figure 1a.

- Figure S1c- according to the literature, the authors should be able to detect the LOS without inner/outer core generated in ∆lpsB mutant – what are the higher molecular weight bands?

- Figure 2f – The color scale is missing.

- Line 147 - The authors should describe the RNA-seq experiment in the text as well as which cells were used.

- Line 152 – It appears that a piece of a sentence is missing

- Line 152-155 - It is unclear in the text why the authors only describe Irf1, Infb1 and Stat2.

- A few references are missing in the results section

Reviewer #3: 1. In the opening sentence of the abstract, virulence typically refers to ability to cause infection by the organism, not molecules such as lipooligosaccharides, so please reword.

2. The manuscript would benefit from additional copyediting throughout.

3. Lines 113-116 appear to be out of place and include figure callouts out of order.

4. Figure 1 A some of the text is too small to read

5. Lines 127-128: Most of the cytokine results are not discussed. It seems odd that the patterns would be so different for some between serum and BALF (e.g. IL-6, KC)—what might these mean for understanding inflammation induced by intact LOS?

6. Why is MIP-2 (murine inflammatory protein 2) shown for Figure 2A with human THP1 macrophages?

7. What are the sequence types of strains shown in Figure 2D?

8. Figure 5: typo-picentannol should be piceatannol

9. Figure 5e: was the difference in pulldown of LOS by CD14 statistically significant?

**********

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Reviewer #1: Yes: Clay D. Jackson-Litteken

Reviewer #2: No

Reviewer #3: No

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PLoS Pathog. 2026 Jul 14;22(7):e1014364. doi: 10.1371/journal.ppat.1014364.r002

Author response to Decision Letter 1

19 Sep 2025

Attachment

Submitted filename: Response to Reviewers 20250919.docx

ppat.1014364.s009.docx^{(987.1KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1014364.r003

Decision Letter 1

Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman

27 Oct 2025

-->PPATHOGENS-D-25-00850R1

Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production

PLOS Pathogens

Dear Dr. Wang,

Please submit your revised manuscript within 60 days Dec 26 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Matthew Wolfgang

Section Editor

PLOS Pathogens

-->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

-->-->Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments :

Two of the original reviewers have re-evaluated this manuscript and remain concerned about several experiments that must be fully addressed in a potential revision. The revised version should experimentally resolve the issue regarding the LOS pattern, as raised by both reviewers. One reviewer noted that “the pulldowns do not appear to show higher affinity for intact LOS to CD14 compared to LBP, as LBP shows ~40 LOS/protein while CD14 shows ~30 LOS/protein.” In the revised version, I expect the authors to provide quantitative measurements of affinity. The issue regarding cytokine responses should also be clarified. If the authors convincingly address these and the additional concerns stated by both reviewers, I would be willing to consider a revised version.

Journal Requirements:

1) Regarding Figure 7, thank you for stating " We've redrawn the figure using the open source materials and uploaded the new Fig 7". Please include in the figure legend a link to the source of the images.

Note: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: (No Response)

Reviewer #3: The authors have addressed some of my previous comments. However, additional information they provide raises new questions on methods and some key experimental comments were not addressed.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #2: - The data obtained from the mouse experiments (Fig. 1J) appear to be the same as in the original manuscript, except normalized to vesicle number rather than 50 µg of protein. Without repeating the mice experiments, the authors should provide biological replicates demonstrating equivalence between the 50 µg protein and the vesicle number normalization, especially since vesicles from the ΔlpsB strain are larger.

- The authors should repeat and include a LOS silver stain to clearly show the truncated LOS used in this study. Currently, it appears as if no LOS is present in the ΔlpsB strain. For reference, see PMID: 38349180 (Fig 7A), where LOS was extracted from ΔlpsB mutant and displayed a shorter LOS band.

Reviewer #3: 1. Strain construction and Table S3-4:

a. First, ∆lpsB mutants are made in multiple strain backgrounds but only one set of primers is included. Are the sequences where the primers bind actually identical in all three strains? That seems unlikely.

b. Ssecond, the description of the methods would not be likely to generate deletion mutants and there are no methods described or primers listed to verify the deletion mutants. Specifically, using 500 bp homology on either side is low efficiency, when combined with selecting for a double crossover in 1 step, it is extremely unlikely to select correct mutants. Second, there are no primers listed that anneal near the lpsB gene but outside of the construct present in the pGEM-lpsB-apraR that would be required to verify the mutant. Thus, the strains used may be single crossovers integrating pGEM-lpsB-apraR into the chromosome.

2. In Figure S1C, there should be detectable LOS without core sugars in an LOS gel, as previously reported (Hood et al., Infect. Immun., 2013). This comment from reviewer 2 remains a concern.

3. It does not seem appropriate to remove all data for other cytokines as the response to reviewers querying about why the data for other cytokines appearing inconsistent.

4. Line 326-327/Figure 5e: the pulldowns do not appear to show higher affinity for intact LOS to CD14 compared to LBP, as the LBP shows ~40 LOS/protein while CD14 shows ~30 LOS/protein. In addition, as reviewers previously raised, this measure of binding is qualitative and additional methods/analyses should be used to measure affinity quantitatively to support the claims of the manuscript.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: - Line 37–38: The current sentence, “Lipooligosaccharides (LOS) with a core region are more pathogenic than those without it; however, the underlying mechanism remains fully elucidated,” is inaccurate, as an LOS molecule itself cannot be described as pathogenic. The sentence should be rephrased in line with line 68–69 which states: “An A. baumannii strain harboring the core region of LOS displayed higher virulence than a strain lacking this core region.” In addition, the phrase “remains fully elucidated” should be corrected to “remains to be fully elucidated.”

- Some figure panels remain too small (e.g., 1B, 1C, 1J), and the labels/writing are difficult to read (e.g., 1D–H).

- Acinetobacter baumannii is not curved, schematics in Fig. 1A, 1I, and S1B should be corrected.

- Fig. 2B: The label “to 1000 ng of LPS” on the y-axis is unclear and should be clarified.

- Fig. 2F: The authors should specify what the scale numbers represent (log₂(FC) or FC).

- Line 181: Please specify the strain/cell type used for RNA-seq.

- Mice were injected with 8.3 × 10¹¹ particles. Given that each bacterium produces ~0.08 vesicles, this corresponds to ~1 × 10¹³ bacteria. Could the authors speculate whether these difference might still be relevant in an infection?

- Fig. 4F: There appear to be issues with the statistical error bars that should be addressed

Reviewer #3: 5. The text in most of the figures is too small

6. The primers to generate complementation vectors are not reported nor any information about pAbYm2 such as antibiotic resistance

7. Table S3 cites reference 48 for the double crossover method, which does not appear to be the correct reference.

8. Antibiotic resistance should be listed/specified for strains used in Table S3 as requested by another reviewer

**********

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Reviewer #2: No

Reviewer #3: No

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Reproducibility:

PLoS Pathog. 2026 Jul 14;22(7):e1014364. doi: 10.1371/journal.ppat.1014364.r004

Author response to Decision Letter 2

11 Mar 2026

Attachment

Submitted filename: Response to Reviewers 0311.docx

ppat.1014364.s010.docx^{(925KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1014364.r005

Decision Letter 2

Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman

5 Apr 2026

-->-->PPATHOGENS-D-25-00850R2

Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production

PLOS Pathogens

Dear Dr. Wang,

Please submit your revised manuscript by Jun 04 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Matthew Wolfgang

Section Editor

PLOS Pathogens-->--> -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

-->-->Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

Overall, the reviewers are pleased with most of the edits and changes in this revised version. However, both reviewers raise concerns about the quality of the SDS-PAGE analysis of LOS (LPS). This issue is not merely cosmetic; the authors need to provide a high-quality silver-stained gel showing the LOS profiles of both the wild-type and mutant strains. One reviewer also suggested including LPS from an additional bacterial species as a reference to aid interpretation. If the authors can improve the SDS-PAGE analysis to the required standard and revise the manuscript in accordance with the reviewers’ comments, the work would be suitable for acceptance.

Journal Requirements:

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: (No Response)

Reviewer #3: Overall, the additional primer and strain construction information clarifies the methods used and mutant generated. However, the LOS gel remains confusing.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #2: The authors have addressed most of my comments.

However, the LOS silver stain remains unsatisfactory, both in terms of quality and interpretation (banding pattern).

Reviewer #3: 1. The LOS gel remains confusing. An A. baumannii lpsB mutant has been reported multiple times by multiple groups to generate LOS band slightly lower than the full length LOS (PMID: 23230287; PMID: 32908144; PMID: 38349180). However, these were all with A. baumannii 17978 so it may be different for Ab908. LOS typically migrates more similarly to ~10 kDa molecular weight. The band labeled LOS in Figure S1D at the dye front is not likely LOS, although it is not clear since there were no controls included here for comparison to e.g. rough type E. coli that runs similarly as A. baumannii LOS.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: (No Response)

Reviewer #3: 1. Some text in figures remains too small, e.g. the axis labels in Figure 1

2. The lpsB deletion design appears to only delete 35 bp in the middle of the gene. This is fine as a partial deletion/insertion, but should likely be described as a disruption or knockout rather than deletion in the results. The description in the methods in Lines 465-468 is appropriate. However, Line 469-470 should be corrected, as the 500 bp homology arms appear to be within the lpsB gene rather than upstream and downstream of the gene.

3. It seems P8 is not depicted in the primer scheme in Figure S1C.

**********

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Reviewer #2: No

Reviewer #3: No

Figure resubmission:

Reproducibility:

PLoS Pathog. 2026 Jul 14;22(7):e1014364. doi: 10.1371/journal.ppat.1014364.r006

Author response to Decision Letter 3

14 May 2026

Attachment

Submitted filename: Response to Reviewers 0509.docx

ppat.1014364.s011.docx^{(1.2MB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1014364.r007

Decision Letter 3

Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman

9 Jun 2026

Dear MD Wang,

We are pleased to inform you that your manuscript 'Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production' has been provisionally accepted for publication in PLOS Pathogens.

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The new LPS figure is much clearer now

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1014364.r008

Acceptance letter

Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman, Matthew Wolfgang, Mario Feldman

Dear MD Wang,

We are delighted to inform you that your manuscript, "Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production," has been formally accepted for publication in PLOS Pathogens.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. 1. The size of membrane vesicles from different strains used in this study.

(DOCX)

ppat.1014364.s001.docx^{(15.5KB, docx)}

S2 Table. 2. Enriched transcription factor (TF) binding motifs in promoters of differentially expressed genes (DEGs).

(DOCX)

ppat.1014364.s002.docx^{(18KB, docx)}

S3 Table. Bacterial isolates used in this study.

(DOCX)

ppat.1014364.s003.docx^{(18.2KB, docx)}

S4 Table. The primers used in this study.

(DOCX)

ppat.1014364.s004.docx^{(18.9KB, docx)}

(TIF)

ppat.1014364.s005.tif^{(774.2KB, tif)}

S2 Fig. RNA sequencing analysis of the gene expression.

(TIF)

ppat.1014364.s006.tif^{(358.1KB, tif)}

S3 Fig. Cytotoxicity of the inhibitors used in this study.

(TIF)

ppat.1014364.s007.tif^{(295.1KB, tif)}

Attachment

Submitted filename: Response to Reviewers 20250919.docx

ppat.1014364.s009.docx^{(987.1KB, docx)}

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Submitted filename: Response to Reviewers 0311.docx

ppat.1014364.s010.docx^{(925KB, docx)}

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Submitted filename: Response to Reviewers 0509.docx

ppat.1014364.s011.docx^{(1.2MB, docx)}

Data Availability Statement

The data that support the findings of this study are openly available in figshare at doi:10.6084/m9.figshare.28028600.

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PERMALINK

Acinetobacter baumannii lipooligosaccharide core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through interferon-β production

Yi-Tzu Lee

Te-Li Chen

Shu-Chen Kuo

Yu-Han Liu

Meng-Chang Lee

Ming-Fen Chuang

Mei-Chei Tan

I-Ming Lee

Ya-Sung Yang

Yung-Chih Wang

Roles

Abstract

Author summary

Introduction

Results

Bacterial strains

0.1 A. baumannii with intact LOS demonstrated higher pathogenicity than those lacking the core region

Fig 1. Acinetobacter baumannii strains with intact lipooligosaccharide (LOS) (Ab908 and Ab908ΔlpsB::lpsB) demonstrated higher virulence than those lacking the core region of LOS (Re-LOS, Ab908ΔlpsB).

MVs containing intact LOS induced higher levels of IFN-β release and greater responsiveness of IFN-β-related genes in macrophages than those containing Re-LOS

Fig 2. Membrane vesicles containing intact lipooligosaccharide (LOS) induced increased IFN-β secretion and higher Infb1 gene expression and its signaling pathway than those lacking the core region of LOS (Re-LOS).

Core region of LOS enhanced IFN-β production through the TLR4-TRIF signaling pathway

Fig 3. The core region of lipooligosaccharide (LOS) is responsible for enhanced IFN-β production.

LOS core region selectively enhances the TRIF-dependent pathway, but without affecting the MyD88-dependent pathway

Fig 4. The core region of lipooligosaccharide (LOS) selectively enhances the TRIF-dependent signaling pathway but not the MyD88-dependent signaling pathway.

Core of LOS enhanced CD14-dependent TLR4 endocytosis

Fig 5. The core region of lipooligosaccharide (LOS) enhances CD14 endocytosis.

IFN-β stimulated by the MVs with intact LOS is associated with increased pathogenicity

Fig 6. Increased IFN-β production was associated with enhanced pathogenicity in lipooligosaccharide(LOS)-treated mice.

Discussion

Fig 7. Acinetobacter baumannii lipooligosaccharide(LOS) core region promotes CD14-dependent TLR4 endocytosis and enhances pathogenicity through IFN-β production.

Conclusion

Materials and methods

Ethics statement

Bacterial strains

Silver staining of LOS

Growth of the isolates

Cell lines and culture conditions

Chemical sources

Animal experiments

Isolation and purification of MVs

Characterization of MVs using a nanoparticle tracking analyzer

Mice pneumonia model

Mice intraperitoneal sepsis model

Enzyme-linked immunosorbent assay for cytokines

RNA extraction and quantitative real-time polymerase chain reaction

RNA sequencing and data analysis

Biological activity

Cytotoxicity of the inhibitors

Western blot analysis

Flow cytometry for TLR4 endocytosis and TLR4/MD-2 dimerization

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Matthew Wolfgang

Mario Feldman

Roles

Author response to Decision Letter 1

Decision Letter 1

Matthew Wolfgang

Mario Feldman

Matthew Wolfgang

Mario Feldman

Roles

Author response to Decision Letter 2

Decision Letter 2

Matthew Wolfgang

Mario Feldman

Matthew Wolfgang

Mario Feldman

Matthew Wolfgang

Mario Feldman

Roles

Author response to Decision Letter 3

Decision Letter 3