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Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi-functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N-deacetylase PgdA and the peptidoglycan O-acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA-dependent and OatA-independent. Protein-protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB-like cell division protein and factors important for escape from the host immune system.

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica . To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD(4) C(2) activity. FlhD(4) C(2) activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of σ(28) activity. We have found that efficient growth-dependent regulation of FlhD(4) C(2) requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhD(4) C(2) activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD(4) C(2) circuit. When FlhD(4) C(2) was not bound to DNA, FliT disrupted the FlhD(4) C(2) complex. Interestingly, when FlhD(4) C(2) was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD(4) C(2) activity by preventing the formation of the FlhD(4) C(2) :DNA complex. Our data would suggest that this level of endogenous regulation of FlhD(4) C(2) activity allows the flagellar system to efficiently respond to external signals.

Background: BA0330 and BA0331 are the only two lipoproteins among 11 known or putative polysaccharide deacetylases from Bacillus anthracis. Results: Both proteins lack deacetylase activity and are important for cell shape maintenance (BA0331) or high salt stress adaptation of the bacterium (BA0330). Conclusion: BA0330 and BA0331 stabilize the cell wall of B. anthracis. Significance: Understanding the mechanisms by which lipoproteins maintain cell wall integrity., Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in Δ fljK Δ fljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis. We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF-alpha secretion in the post-transplantation strain.

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.

Members of genus Burkholderia include opportunistic Gram-negative bacteria that are responsible for serious infections in immunocompromised and cystic fibrosis (CF) patients. The Burkholderia cepacia complex is a group of microorganisms composed of at least nine closely related genomovars. Among these, B. cenocepacia is widely recognized to cause epidemics associated with excessive mortality. Species that belong to this strain are problematic CF pathogens because of their high resistance to antibiotics, which makes respiratory infections difficult to treat and impossible to eradicate. Infection by these bacteria is associated with higher mortality in CF and poor outcomes following lung transplantation. One virulence factor contributing to this is the pro-inflammatory lipopolysaccharide (LPS) molecules. Thus, the knowledge of the lipopolysaccharide structure is an essential prerequisite to the understanding of the molecular mechanisms involved in the inflammatory process. Such data are instrumental in aiding the design of antimicrobial compounds and for developing therapeutic strategies against the inflammatory cascade. In particular, defining the structure of the LPS from B. cenocepacia ET-12 clone LMG 16656 (also known as J2315) is extremely important given the recent completion of the sequencing project at the Sanger Centre using this specific strain. In this paper we address this issue by defining the pro-inflammatory activity of the pure lipopolysaccharide, and by describing its full primary structure. The activity of the lipopolysaccharide was tested as a stimulant in human myelomonocytic U937 cells. The structural analysis was carried out by compositional analysis, mass spectrometry and 2D NMR spectroscopy on the intact lipooligosacchride (LOS) and its fragments, which were obtained by selective chemical degradations.