The peptidoglycan-associated lipoprotein gene mutant elicits robust immunological defense in mice against Salmonella enteritidis.

Background: Salmonella enteritidis (S. enteritidis), a zoonotic pathogen with a broad host range, presents a substantial threat to global public health safety. Vaccination stands as an effective strategy for the prevention and control of S. enteritidis infection, highlighting an immediate clinical need for the creation of safe and efficient attenuated live vaccines. Methods: In this study, a S. enteritidis peptidoglycan-associated lipoprotein (pal) gene deletion strain (Δpal), was constructed. To assess its virulence, we conducted experiments on biofilm formation capability, motility, as well as cell and mouse infection. Subsequently, we evaluated the immune-protective effect of Δpal. Results: It was discovered that deletion of the pal gene reduced the biofilm formation capability and motility of S. enteritidis. Cell infection experiments revealed that the Δpal strain exhibited significantly decreased abilities in invasion, adhesion, and intracellular survival, with downregulation of virulence gene expression, including mgtC, invH, spvB, sipA, sipB, ssaV, csgA, and pipB. Mouse infection experiments showed that the LD50 of Δpal increased by 104 times, and its colonization ability in mouse tissue organs was significantly reduced. The results indicated that the pal gene severely affected the virulence of S. enteritidis. Further, immunogenicity evaluation of Δpal showed a significant enhancement in the lymphocyte transformation proliferation capability of immunized mice, producing high titers of specific IgG and IgA, suggesting that Δpal possesses good immunogenicity. Challenge protection tests demonstrated that the strain could provide 100% immune protection against wild-type strains in mice. Discussion: This study proves that the pal gene influences the virulence of S. enteritidis, and Δpal could serve as a candidate strain for attenuated live vaccines, laying the foundation for the development of attenuated live vaccines against Salmonella.

Direct interaction between fd phage pilot protein pIII and the TolQ-TolR proton-dependent motor provides new insights into the import of filamentous phages.

Filamentous phages are one of the simplest examples of viruses with a protein capsid that protects a circular single-stranded DNA genome. The infection is very specific, nonlytic, and can strongly affect the physiology or provide new pathogenic factors to its bacterial host. The infection process is proposed to rely on a pore-forming mechanism similar to that of certain nonenveloped eukaryotic viruses. The Ff coliphages (including M13, fd, and f1) have been intensively studied and were used to establish the sequence of events taking place for efficient crossing of the host envelope structure. However, the mechanism involved in the penetration of the cell inner membrane is not well understood. Here, we identify new host players involved in the phage translocation mechanism. Interaction studies by a combination of in vivo biochemical methods demonstrate that the adhesion protein pIII located at the tip of the phage binds to TolQ and TolR, two proteins that form a conserved proton-dependent molecular motor in the inner membrane of the host cell. Moreover, in vivo cysteine cross-linking studies reveal that the interactions between the pIII and TolQ or TolR occur between their transmembrane helix domains and may be responding to the proton motive force status of the cell. These results allow us to propose a model for the late stage of filamentous phage translocation mediated by multiple interactions with each individual component of the host TolQRA complex.

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology, antibiotic resistance and virulence.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most successful human pathogens worldwide and responsible for hospital-acquired infections. This is due to its outstanding potential to adapt to very different environments, to persist in the human host and most important, its ability to develop multidrug resistance. Our combined approach of genomic and phenotypic analyses led to the identification of the envelope spanning Tol-Pal system in A. baumannii. We found that the deletion of the tolQ, tolR, tolA, tolB, and pal genes affects cell morphology and increases antibiotic sensitivity, such as the ∆tol-pal mutant exhibits a significantly increased gentamicin and bacitracin sensitivity. Furthermore, Galleria mellonella caterpillar killing assays revealed that the ∆tol-pal mutant exhibits a decreased killing phenotype. Taken together, our findings suggest that the Tol-Pal system is important for cell morphology, antibiotic resistance, and virulence of A. baumannii.

The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate.

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol-Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol-Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol-Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol-Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol-Pal system.

Timing of TolA and TolQ Recruitment at the Septum Depends on the Functionality of the Tol-Pal System.

Efficient cell division of Gram-negative bacteria requires the presence of the Tol-Pal system to coordinate outer membrane (OM) invagination with inner membrane invagination (IM) and peptidoglycan (PG) remodeling. The Tol-Pal system is a trans-envelope complex that connects the three layers of the cell envelope through an energy-dependent process. It is composed of the three IM proteins, TolA, TolQ and TolR, the periplasmic protein TolB and the OM lipoprotein Pal. The proteins of the Tol-Pal system are dynamically recruited to the cell septum during cell division. TolA, the central hub of the Tol-Pal system, has three domains: a transmembrane helix (TolA1), a long second helical periplasmic domain (TolA2) and a C-terminal globular domain (TolA3). The TolQR complex uses the PMF to energize TolA, allowing its cyclic interaction via TolA3 with the OM TolB-Pal complex. Here, we confirm that TolA2 is sufficient to address TolA to the site of constriction, whereas TolA1 is recruited by TolQ. Analysis of the protein localization as function of the bacterial cell age revealed that TolA and TolQ localize earlier at midcell in the absence of the other Tol-Pal proteins. These data suggest that TolA and TolQ are delayed from their septal recruitment by the multiple interactions of TolA with TolB-Pal in the cell envelope providing a new example of temporal regulation of proteins recruitment at the septum.

Pal depletion results in hypervesiculation and affects cell morphology and outer-membrane lipid asymmetry in bordetellae.

Current vaccines against Bordetella pertussis do not prevent colonization and transmission of the bacteria, and vaccine-induced immunity wanes rapidly. Besides, efficacy of vaccines for Bordetella bronchiseptica remains unclear. Novel vaccines could be based on outer-membrane vesicles (OMVs), but vesiculation of bordetellae needs to be increased for cost-effective vaccine production. Here, we focused on increasing OMV production by reducing the anchoring of the outer membrane to the peptidoglycan layer. Inactivation of rmpM, tolR, and pal failed, presumably because their products are essential in bordetellae. Conditional pal mutants were constructed, which were hypervesiculating under Pal-depletion conditions. SDS-PAGE and Western blot analyses showed that the protein composition of OMVs produced under Pal-depletion conditions resembled that of the outer membrane but differed from that of OMVs released by the wild type. Pal depletion affected the cell morphology and appeared to increase the amounts of cell-surface-exposed phospholipids, possibly reflecting a role for the Tol-Pal system in retrograde phospholipid transport. We also identified additional lipoproteins in bordetellae with a putative peptidoglycan-anchoring domain. However, their inactivation did not influence OMV production. We conclude that the conditional pal mutants could be valuable for the development of OMV-based vaccines.

The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis.

The Tol-Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in E. coli, and subsequently been identified in many other bacterial genera. However, the function of the Tol-Pal system in Salmonella Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of S. Choleraesuis. We found that mutations in tolA, tolB, and tolR caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased S. Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition, tolA, tolB, and tolR mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from ΔtolB efficiently enhanced murine viability in contrast to OMVs from the wild-type S. Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the S. Choleraesuis Tol-Pal system.

Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 ΔtolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and ΔtolA OMVs, where ΔtolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and ΔtolA OMVs confirmed that ΔtolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli.

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host's existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA.IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

Tol Energy-Driven Localization of Pal and Anchoring to the Peptidoglycan Promote Outer-Membrane Constriction.

During cell division, gram-negative bacteria must coordinate inner-membrane invagination, peptidoglycan synthesis and cleavage and outer-membrane (OM) constriction. The OM constriction remains largely enigmatic, and the nature of this process, passive or active, is under debate. The proton-motive force-dependent Tol-Pal system performs a network of interactions within these three compartments. Here we confirm that the trans-envelope Tol-Pal complex accumulates at constriction site in Escherichia coli. We show that the inner-membrane complex composed of TolA, TolQ and TolR recruits the OM complex TolB-Pal to the septum, in an energy-dependent process. Pal recruitment then allows its binding to peptidoglycan and subsequently OM constriction. Our results provide evidence that the constriction of the OM is an energized process.