Abstract
Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.
Introduction
Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative coccobacillus and human-restricted opportunistic pathogen (). NTHi causes a wide range of mucosal infections in the upper and lower respiratory tract. This includes acute otitis media and exacerbations in patients with asthma and chronic obstructive pulmonary disease (; Van Eldere et al., 2014; Su et al., 2018; ). Several recent reports have also suggested NTHi in causing invasive diseases (Van Eldere et al., 2014; ; Tonnessen et al., 2022).
NTHi possesses a plethora of mechanisms to colonize while evading the host immune system for establishment of subsequent infection. The pathogen expresses a variety of virulence factors involved in: (i) acquisition of complement regulators (i.e., C4b-binding protein (C4BP), vitronectin and factor H (FH)) to suppress complement-mediated killing, and (ii) adherence to host cell receptors (i.e., CEACAMs and ICAM-1) and extracellular matrix proteins (i.e. fibronectin, laminin, vitronectin, and collagen IV) for colonization at the airway mucosa (; ; ; Ronander et al., 2009; ; Su et al., 2013; ; Rosadini et al., 2014; Su et al., 2016; Su et al., 2019; Thofte et al., 2021). NTHi also occasionally harbours mutated penicillin-binding proteins (PBPs), produces β-lactamases and forms biofilm for antimicrobial resistance (Thegerstrom et al., 2018; ; Shin et al., 2021).
One of the most studied virulence factors of NTHi is the major outer membrane (OM) protein 5 (P5). NTHi P5 is a ~35 kDa outer membrane protein (Omp) A family protein. It is composed of two main domains: (i) conserved N-terminal membrane-embedded β-barrel transmembrane domains with four highly variable and immunogenic extracellular surface loops (namely loop 1-4) and (ii) a conserved periplasmic C-terminal domain (CTD) (Figure 1A) (Webb and Cripps, 1998; ). The multifunctional surface loops of P5 contribute to NTHi adherence by binding to mucin, ICAM-1 and CEACAM1; and serum resistance by acquisition of C4BP and FH (Reddy et al., 1996; ; ; ; Rosadini et al., 2014; Thofte et al., 2021). Even though the molecular structure of P5 has been extensively investigated in the aspect of host-pathogen interactions with focus on the extracellular loops, there is a paucity of information regarding the impact of periplasmic CTD of P5 in NTHi physiology and pathogenesis. Here we report a novel role of P5 in maintaining bacterial OM integrity and membrane protein composition important for NTHi-host interactions. Our findings provide useful knowledge regarding the direct and indirect role of P5 in the pathogenesis of NTHi.
Figure 1
Materials and methods
Bacterial strains and eukaryotic cells
Escherichia coli DH5α (Stratagene, Santa Clara, CA) and BL21 (DE3) (Novagen, Darmstadt, Germany) were used as cloning and protein expression hosts, respectively. The genetic information and cultured conditions for E. coli, NTHi 3655 and the isogenic mutants are summarized in Supporting Table S1. Construction of p5CTD-knockout (NTHi 3655Δp5CTD) and p5-transcomplementation (NTHi 3655Δp5::p5) mutants is described in Supporting Figure S1 and Table S2. NTHi mutants were verified for surface display of P5 by flow cytometry using rabbit anti-peptide P5Loop3 polyclonal antibodies (pAbs) (Genscript, Piscataway, NJ) (Thofte et al., 2021). The human alveolar epithelial cell line A549 (ATCC CCL-185™) (American Type Culture Collection (ATCC), Manassas, VA) was maintained in F12 medium supplemented with 2 mM L-glutamine, and 10% fetal calf serum (FCS) (Gibco, Life Technologies, Carlsbad, CA).
Isolation of membrane fractions and outer membrane vesicles
Bacterial membrane fractions and OMVs were isolated from mid and late-log phase cultures, respectively as described (Su et al., 2019; ). Briefly, membrane fractions were pelleted from the supernatant of sonicated bacterial lysate at 150,000×g at 4°C for 1 hour. Bacterial lysates or membrane fractions were separated on 12% SDS-PAGE followed by western blotting. Anti-peptide P5Loop3 pAb and horse radish peroxidase (HRP)-conjugated donkey anti-rabbit pAb (Abcam, Cambridge, UK) were used to detect P5 on the blots (Thofte et al., 2021). The OM fractions (from three replicated preparations) were subjected to proteomic analysis (Proteomics Biomedicum, Karolinska Institutet, Sweden) (Table S3 and Supporting Methodology). The yield and particle size of purified OMVs were determined by nanoparticle tracking analysis (NTA) with a NanoSight NS300 (Malvern Panalytical, Malvern, UK).
Recombinant protein expression
DNA fragment encoding P5CTD (M233 to K359) was amplified from plasmid P53655-pET16b (Thofte et al., 2021) using specific primers (Table S2) and cloned into the expression vector pET26(b)+ (Novagen). His-tagged recombinant protein was expressed in E. coli BL21 (DE3) with 1 mM IPTG induction and purified as described (Su et al., 2013).
Peptidoglycan-binding assay
The assay was performed as described (; ). Briefly, 3 µM of His-tagged recombinant protein was incubated with 100 µg of peptidoglycan from E. coli K12 (Invivogen, Toulouse, France) in 100 µl of ice-cold binding buffer (10 mM Tris-Cl, 10 mM MgCl2, 50 mM NaCl, pH6.8) at 4°C for 16 hours. Mixtures were pelleted at 350,000×g at 4°C for 1 hour. The supernatants were collected as unbound protein; the pellets were washed twice with the ice-cold binding buffer. Finally, the pellets were dissolved in 2% SDS. The supernatants and pellets were analysed on 12% SDS-PAGE and western blotting. His-tagged proteins were detected with HRP-conjugated anti-His pAb (Abcam).
Adherence assay
Bacterial adherence to human epithelial cells was performed as described (Su et al., 2016). Briefly, cells grown to 85% confluency were infected with mid-log phase bacteria (OD600 = 0.5) to achieve a multiplicity of infection (MOI) of 100, at 37°C and 5% CO2 in F12 media without FCS for 30 min. Cells were washed with PBS and detached with trypsin-EDTA (Sigma-Aldrich, St. Louis, MO). Cell solutions were plated on chocolate agar and incubated at 37°C and 5% CO2 for 16 hours to quantify adherent bacteria in colony-forming units (CFU).
Fibronectin-binding assay
The assay was performed by flow cytometry as described (Su et al., 2019). Mid-log phase (OD600 = 0.5) bacteria (5×107 CFU) were incubated with indicated concentrations of human fibronectin (Sigma-Aldrich) in 100 µl of 1% BSA at 37°C and 5% CO2 for 1 hour. Samples were washed and pelleted by centrifugation. Bacteria-bound fibronectin was detected with rabbit anti-human fibronectin pAb (Dako, Glostrup, Denmark) and FITC-conjugated swine anti-rabbit pAb (Dako). Samples were analysed in a BD FACSVerse™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ).
Serum resistance assay
The assay was performed as described (Su et al., 2013). Suspension of mid-log phase (OD600 = 0.5) bacteria (1.5×103 CFU) in 150 µl of DVBS-BSA buffer was incubated with 5% normal human serum (NHS) for indicated time points at 37°C. Samples were plated on chocolate agar and incubated at 37°C and 5% CO2 for 16 hours.
Hyperosmotic assay
The assay was performed as described (). Bacteria grown to mid-log phase (OD600 = 0.5) were pelleted, washed, and resuspended in PBS to OD60 = 1.0. Bacterial suspensions were 10-fold serially diluted in PBS to generate 104-109 CFU/ml, and 2 µl from each dilution was spotted on chocolate agar supplemented with 50 mM or 100 mM NaCl. Plates were incubated at 37°C and 5% CO2 for 16 hours.
Broth microdilution
Broth microdilution (BMD) was performed in three independently repeated-experiments according to ISO 20776-1 on custom freeze-dried Sensititre plates (Thermo Scientific, Basingstoke, UK) using the EUCAST recommended broth for fastidious organisms (Mueller Hinton Fastidious (MH-F) broth) to estimate minimal inhibitory concentrations (MICs) of antibiotics. MIC endpoints after 16-20 hours of incubation at 35 ± 1°C were visually evaluated according to ISO and EUCAST recommendations.
Statistical analyses
We used GraphPad Prism® 9.0 (GraphPad Software, La Jolla, CA) for statistical analyses. One- or two-way ANOVA tests were used as indicated. Differences were considered statistically significant at P ≤ 0.05.
Results
The periplasmic C-terminal domain of P5 binds peptidoglycan
Based on bioinformatic analysis, P5 of NTHi 3655 belongs to the OmpA family of proteins (InterPro entry number: IPR002368) (Figures 1A, S2). An OmpA-like domain (IPR006665) was found at the C-terminal domain (CTD) of P5 (P5CTD) that was located between residue M233 and K359. The P5CTD shared 20.6-56.2% of sequence similarity (34.6-67.2% identity) with some peptidoglycan-associated lipoproteins (Pal) and the C-terminus of other OmpA proteins that are known to have an OmpA-like domain, and experimentally bind peptidoglycan (Figure 1B). While the protein sequence homology of CTDs among OmpA family proteins is generally poor, two key residues (i.e., D283 and R298) that were crucial for peptidoglycan binding of OmpA in Acinetobacter baumannii and E. coli were also found on the P5CTD (; Samsudin et al., 2016).
The OmpA-like domain has a peptidoglycan-binding property via non-covalent interactions (; ; Samsudin et al., 2016). We therefore hypothesize that P5 also might function in an OmpA-like domain-dependent peptidoglycan binding. We observed that purified His-tagged P5CTD formed a complex with peptidoglycan, which was almost comparable to His-tagged P6 (positive control) (Figures 1C, D). P6 is a Pal with an OmpA-like domain and is known to bind peptidoglycan in NTHi (; ). P6 does not have a transmembrane beta-barrel domain as seen in OmpA. The negative control, His-tagged P4 of NTHi 3655 (; Su et al., 2016) did not bind peptidoglycan. Taken together, our data demonstrates that the P5CTD has a peptidoglycan-binding activity as predicted by in silico analysis thus may play a role in the OM integrity of NTHi.
Deletion of P5 leads to altered membrane proteome composition in NTHi
The OmpA-like domain serves as an anchor between the OM layer and the peptidoglycan. The interaction is crucial for OM-cell wall stability, and the assembly of membrane and cell wall proteins (; ; ; ; ; Samsudin et al., 2016; ; ). Since we showed that P5CTD of NTHi 3655 has peptidoglycan-binding activity, we hypothesized a possible role for P5 in the distribution of NTHi periplasmic and membrane protein (membrane proteome). To this end, we generated NTHi 3655Δp5CTD that expressed CTD-deleted P5, and a P5-transcomplemented mutant (NTHi 3655Δp5::p5). In addition, NTHi 3655Δp5 that has a full deletion of P5 was included as a control for comparison to the deletion of P5CTD (Thofte et al., 2021).
Comparative SDS-PAGE of bacterial whole cell lysates and membrane fractions revealed some differences in the intensity of a few protein bands among the NTHi 3655 wild-type and mutants (Figures 2A, B). NTHi 3655Δp5CTD and Δp5 exhibited a roughly similar pattern of protein profiling; while a simpler similarity of protein profiling pattern was observed between the wild-type and Δp5::p5. In western blotting, wild-type P5 and CTD-truncated P5 were detected by anti-P5Loop3 pAb in NTHi 3655 and Δp5CTD, respectively. This indicated that the deletion of CTD did not hamper protein expression of the remaining extracellular-loops and the transmembrane parts of P5 in NTHi 3655Δp5CTD. Any corresponding signal was not detected in NTHi 3655Δp5 that was used as a negative control. Interestingly, we observed reduced detection of P5 on the surface of Δp5CTD compared to the wild-type by flow cytometry analyses (Figure 2C). The signal was, however, higher than the P5-knockout mutant, suggesting partial or reduced exposure of P5 extracellular structures on the surface of Δp5CTD. Unexpectedly, Δp5::p5 also exhibited partial detection of P5 by flow cytometry compared to the wild-type. It is currently unclear what causes the partial exposure of P5 extracellular structures in Δp5::p5; it is not known if there are any spontaneous genetic mutations that could affect periplasmic post-translational modifications essential for OM insertion of P5 (; Sklar et al., 2007). Since the transcomplemented strain could not fully restore the surface expression of P5, this strain was excluded from downstream experiments.
Figure 2
To globally understand how P5 is involved in regulating membrane protein organization, we primarily focused on analysing the proteomic composition of the membrane from Δp5 and compared with the wild-type strain. The bacterial membrane preparations used in this study contained mainly OM and periplasmic proteins. Based on the ranking of relative abundance, virulence factors such as major adhesins (i.e., High molecular weight adhesin (HMW) and Haemophilus adhesin protein (Hap)) and IgA protease were less represented while P2, PD, PF and PE were more abundant in the P5-knockout mutant, with respect to the wild-type strain (Table S3) (). We also noticed that periplasmic chaperones (SurA, Omp26 and DegP) and Bam complex subunit proteins (BamA, C, D and E) that are essential for proper folding and assembly of OM proteins, respectively, were less abundant in the mutant (; ; ).
In conclusion, our results suggest that P5 has an influence on the proteomic composition of OM and periplasm in NTHi. P5 may help to maintain optimal interactions between the OM layer and the periplasmic peptidoglycan network that is crucial for cell envelope integrity and protein assembly.
NTHi expressing CTD-deleted P5 exhibits reduced adherence to pulmonary epithelial cells and extracellular matrix protein, and is serum sensitive
The aberrant membrane proteome composition in NTHi upon the deletion of P5 is likely to affect some cellular functions that are crucial for NTHi pathogenesis (; ). Previous studies have shown that P5 deletion in different NTHi isolates results in defective adherence to host cells and increased serum sensitivity compared to the P5-expressing NTHi (; Rosadini et al., 2014; ; Wong et al., 2016; Thofte et al., 2021).
Considering that NTHi 3655Δp5CTD has a reduced exposure of P5 on the surface while showing almost similar membrane profiling on SDS-PAGE as the NTHi 3655Δp5, we wanted to investigate whether the P5CTD truncation could also affect bacterial pathogenicity in a similar fashion as when P5 was fully deleted.
We therefore tested the bacteria by in vitro functional assays related to adherence to host epithelial cells and evasion of host immune system since these two mechanisms are crucial for NTHi persistence to establish infection in the respiratory tract (). The adherence of NTHi 3655Δp5CTD to the lower airway A549 epithelial cells and extracellular matrix protein (fibronectin) was almost comparable to NTHi 3655Δp5, which were significantly (P ≤ 0.001) reduced compared to the NTHi 3655 wild-type (Figures 3A, B). When exposed to 5% NHS for 10 min, deletion of P5CTD rendered the Δp5CTD mutant to be more serum sensitive compared to the wild-type (Figure 3C). However, the Δp5CTD mutant survived better (53% of survival) than the NTHi 3655Δp5 which was almost fully killed (1.9% survival) when exposed to NHS. The data confirm that deletion of P5CTD could impair NTHi pathogenicity similarly as the full deletion of P5. We concluded that P5CTD has an important impact on bacterial pathogenesis.
Figure 3
NTHi 3655Δp5CTD and Δp5 mutants are hypervesiculating and have compromised membrane integrity
Gram-negative bacteria with genetic knockouts of OmpA-like domain superfamily proteins (InterPro: IPR036737) are unable to establish bonds between the OM and peptidoglycan, leading to increased release of OMVs (). To evaluate whether the deletion of either CTD or full length P5 could affect the vesiculation of NTHi, naturally released OMVs were purified from the late-log phase bacterial culture and normalized by cell density. Both NTHi 3655Δp5CTD and Δp5 produced extensive amounts of OMVs as compared to the NTHi 3655 (Figure 3D). Interestingly, NTHi 3655Δp5 was the most hypervesiculating strain, producing almost 2-fold more OMVs than the Δp5CTD mutant (Figure 3D).
The increased OMV production suggested an OM instability among the Δp5CTD and Δp5 mutants. To test this hypothesis, we performed an osmotic shock assay by growing bacteria on chocolate agar supplemented with different NaCl concentrations. When cultured on chocolate agar without additional NaCl or supplemented with 50 mM NaCl, similar to the wild-type, NTHi 3655Δp5CTD and Δp5 mutants did not show any growth defect (Figure 3E). However, at a higher NaCl concentration (100 mM), NTHi 3655Δp5CTD and Δp5 mutants were equally sensitive to the high salt hence showed defective growth, compared to the wild-type. In line with these results, the Δp5CTD and Δp5 mutants equally displayed a 2- and 4-fold susceptibility to β-lactam antibiotics ampicillin (MIC=0.12 µg/ml) and imipenem (0.12 µg/ml), respectively compared to NTHi 3655 (MIC=ampicillin 0.25 µg/ml; imipenem 0.5 µg/ml). Collectively, our data indicate that the CTD of P5 is important in maintaining OM stability and integrity, hence bacterial vesiculation and beta-lactam antibiotics resistance.
Discussion
The OmpA-like domain has been extensively studied at the molecular level for OmpA homologs in several pathogens including E. coli, A. baumannii, Vibrio cholerae, Neisseria meningitidis, and Salmonella enterica. It was shown to contribute to cell envelope stability by forming non-covalent linkages between the OM and peptidoglycan network (; ; ; ; Samsudin et al., 2016). In contrast, the relevance of OmpA-like domain of P5 in NTHi cell envelope physiology hence pathogenesis has not yet been addressed. Bioinformatics analysis in combination with in vitro peptidoglycan-binding assays revealed the presence of the OmpA-like domain at the C-terminal of P5 that was proven to be active in forming a complex with the peptidoglycan (Figure 1). While the mechanism of the P5CTD-peptidoglycan interaction remains to be experimentally elucidated, we postulate that residues D283 and R298 of P5 might interact with the mDAP residue from the peptidoglycan. Ten residues (A313-A343) from the peptidoglycan-bound P5CTD will then interact with the lipid group of the inner leaflet of OM. This in turn stabilizes the P5CTD for stronger interaction with the peptidoglycan while enabling peptidoglycan to further interact with the OM. In general, P5 functions as an anchor to tether the OM to the underlying peptidoglycan, ensuring optimal periplasmic distance between the outer and inner membrane. This architecture stabilizes the OM on the NTHi cell envelope (Figure 4), as previously described for the OmpA-peptidoglycan models from A. baumannii and E. coli (; Samsudin et al., 2016). Our postulation is based on two rationales: (i) the annotation of D283 and R298 in P5CTD that are also the key residues in A. baumannii OmpA for peptidoglycan binding; and (ii) the sequence similarity between the A313-A343 of P5CTD (71% similarity; 74.2% identity) and the OM’s inner leaflet-binding domain of E. coli OmpA (Figure S2).
Figure 4
To the best of our knowledge, this is the first report in deciphering the dynamics of the P5-dependent NTHi membrane proteome. Interestingly, the aberrant protein compositions in the bacterial membrane in response to P5 deletion or truncation of P5CTD (Figure 2 and Table S3) did not alter bacterial cell morphology neither defect bacterial growth (Figure S3). Instead, it was more associated with a reduced pathogenic phenotype as seen in NTHi 3655Δp5 and to some extent, NTHi 3655Δp5CTD (Figure 3). In previous reports, deletion of proteins involved in the OM and cell wall physiology (i.e., VacJ, YcrB and EnvC) of NTHi also resulted in altered membrane proteome, causing the vacJ, ycrB and envC-knockout mutants to be serum sensitive, attenuated in adherence and biofilm formation, and are hypervesiculating (; Roier et al., 2016). We speculate that the reduced appearance of periplasmic chaperones (SurA, Omp26, and DegP) and BamACDE complex in the P5-knockout mutant could probably impair the maturation, and degradation of some misfolded proteins. This ultimately led to the accumulation of some OM proteins in the periplasm or the OM. This might explain the increased abundance of P2 in NTHi 3655Δp5, which was probably in an unfolded/misfolded state. The impaired membrane protein assembly machineries could also affect the presentation of autotransporters (i.e., Hap and HMW), or β-barrel membrane protein like CTD-deleted P5 on the surface of NTHi 3655Δp5 and NTHi 3655Δp5CTD, respectively. Further experimental investigation is, however, needed to unveil the molecular mechanism on how P5 or P5CTD deletion could globally alter the expression level, and protein folding state of certain membrane proteins.
The proteomic characterization of the bacterial membrane of NTHi 3655Δp5CTD is currently ongoing, and the results will be part of a follow-up study. Therefore, besides the reduced exposure of surface P5 extracellular loops, we are currently unable to define the exact additional factors that might contribute to the NTHi 3655Δp5CTD altered pathogenic phenotypes (Figure 3). We speculated that the partial exposure of P5 extracellular structures on the surface NTHi 3655Δp5CTD could be associated with the deletion of CTD of P5 in addition to other unknown factors, suggesting that the P5CTD might play a role in the OM insertion of P5. In E. coli, OM assembly of OmpA partly depends on the periplasmic post-translational modifications on the CTD, including hydrophobicity modification by oligo-(R)-3-hydroxybutyrates and disulphide bond formation between residues C311 and C323 (Patel et al., 2009; Qu et al., 2009; ). Nevertheless, the proteomic data of NTHi 3655Δp5 will be used as a reference for NTHi 3655Δp5CTD in this study, considering their almost similar protein profiling on SDS-PAGE (Figure 2). In addition to the absence of P5 on the bacterial surface, the decreased abundance of Hap and HMW might partly contribute to the reduced adherence of NTHi 3655Δp5, and probably also for NTHi 3655Δp5CTD, to the host epithelial cells (Figures 2, 3) (; ; ; ; ). However, the reduced fibronectin binding among the mutants could be mainly attributed to the decreased density of Hap (Figure 3). This is because Hap has been known as the major fibronectin-binding protein in NTHi but not the P5 (; Su et al., 2016). Moreover, the presence of P4, PF and PE did not improve the bacterial adherence, fibronectin binding, and serum resistance of NTHi 3655Δp5CTD and Δp5 (; Ronander et al., 2009; Su et al., 2013; Su et al., 2016; Su et al., 2019). The reduced exposure of P5 loops on the bacterial surface of NTHi 3655Δp5CTD might reduced the recruitment of C4BP and FH hence increased sensitivity to serum killing (Figure 3), as previoulsy reported for P5-knockout mutants (; Rosadini et al., 2014; Thofte et al., 2021).
Lipooligosaccharide (LOS) biosynthesis and assembly are related to protein assembly on the OM (Ried et al., 1990; ; Spahich et al., 2014). Hence, full P5 deletion might also potentially affect the assembly of LOS and the exposure of surface immunogenic antigens, however, with greater impact than the partial deletion of P5. This might cause the LOS and surface antigens of NTHi3655Δp5 to be more accessible to C-reactive protein or antibodies from NHS (; Roier et al., 2012; ), hence more complement-mediated killing of NTHi 3655Δp5 than the Δp5CTD. This postulation remains, however, to be experimentally elucidated.
The loss of OM integrity was most likely caused by the absence of P5CTD in both NTHi 3655Δp5CTD and Δp5 mutants, leading to hypervesiculation while defective in protecting bacteria from a hyperosmotic environment (Figures 3, 4) (; ). Despite P6 being present in Δp5 and probably also in Δp5CTD, with equal abundance as in the wild-type (Table S3), P6 alone was not sufficient to maintain the linkage between the OM and peptidoglycan without the P5CTD. In E. coli, co-interactions among Braun’s lipoprotein, OmpA and peptidoglycan are essential in OM stability and integrity to protect bacterial cells from high internal osmotic pressure (Samsudin et al., 2016; Samsudin et al., 2017). Decreased expression of PBP has also been associated with bacterial resistance against β-lactam antibiotics (Reygaert, 2018; ). It is possible that the increased abundance of PBP3 and the compromised OM might promote killing of NTHi 3655Δp5CTD and Δp5 by ampicillin and imipenem (; ). A mutant of A. baumannii expressing OmpA without an OmpA-like domain (homolog of P5CTD) was also more susceptible to imipenem compared to the wild-type strain ().
In conclusion, our study sheds light upon the novel role of P5 which is important in regulating NTHi membrane integrity via the OmpA-like domain. The P5CTD has an impact on the distribution of the OM proteins including a plethora of virulence factors, hence NTHi pathogenesis in general.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Author contributions
YCS, MK and KR coordinated the manuscript. YCS and KR drafted the manuscript. AV and RZ performed proteomic analysis. MK and FJ constructed mutants and analysed bacterial protein profiles on SDS-PAGE. MK and OT worked on OMVs preparation and analysis. MS performed bacterial fibronectin-binding assays. YCS and MK carried out bacterial adherence and hyperosmotic assays. MJ conducted surface detection of P5. SJ did bacterial serum resistance assay and growth curve study. LS analysed bacterial cell morphology on TEM. and BMD was done by EM. All authors contributed to the article and approved the submitted version.
Acknowledgments
This work was supported by the Knut and Alice Wallenberg Foundation (KR; 2018.0318), Anna and Edwin Berger Foundation (KR), Swedish Heart Lung Foundation (KR; #20180401, www.hjart-lungfonden.se), the Royal Physiographical Society in Lund (to MK, SJ, OT, MJ, MS; Forssman’s Foundation), the Skåne County Council’s research and development foundation (KR), and Swedish Research Council (KR; #2019-01053, www.vr.se). The electron microscopy was conducted at the Umeå Centre for Electron Microscopy. Proteomic analysis was performed in Proteomics Biomedicum, Karolinska Institutet (Stockholm, Sweden).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2023.1085908/full#supplementary-material
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Summary
Keywords
adherence, extracellular matrix, NTHI, P5, peptidoglycan, serum resistance, virulence
Citation
Su Y-C, Kadari M, Straw ML, Janoušková M, Jonsson S, Thofte O, Jalalvand F, Matuschek E, Sandblad L, Végvári Á, Zubarev RA and Riesbeck K (2023) Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host. Front. Cell. Infect. Microbiol. 13:1085908. doi: 10.3389/fcimb.2023.1085908
Received
31 October 2022
Accepted
10 April 2023
Published
26 May 2023
Volume
13 - 2023
Edited by
Jesús A. Arenas, University of Zaragoza, Spain
Reviewed by
Natalia Korotkova, University of Kentucky, United States; Erwin Bohn, University of Tübingen, Germany; Jeremy Derrick, The University of Manchester, United Kingdom
Updates
Copyright
© 2023 Su, Kadari, Straw, Janoušková, Jonsson, Thofte, Jalalvand, Matuschek, Sandblad, Végvári, Zubarev and Riesbeck.
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*Correspondence: Kristian Riesbeck, kristian.riesbeck@med.lu.se
†These authors have contributed equally to this work
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