Nitrosative stress defences of the enterohepatic pathogenic bacterium Helicobacter pullorum

Helicobacter pullorum is an avian bacterium that causes gastroenteritis, intestinal bowel and hepatobiliary diseases in humans. Although H. pullorum has been shown to activate the mammalian innate immunity with release of nitric oxide (NO), the proteins that afford protection against NO and reactive nitrogen species (RNS) remain unknown. Here several protein candidates of H. pullorum, namely a truncated (TrHb) and a single domain haemoglobin (SdHb), and three peroxiredoxin-like proteins (Prx1, Prx2 and Prx3) were investigated. We report that the two haemoglobin genes are induced by RNS, and that SdHb confers resistance to nitrosative stress both in vitro and in macrophages. For peroxiredoxins, the prx2 and prx3 expression is enhanced by peroxynitrite and hydrogen peroxide, respectively. Mutation of prx1 does not alter the resistance to these stresses, while the single ∆prx2 and double ∆prx1∆prx2 mutants have decreased viability. To corroborate the physiological data, the biochemical analysis of the five recombinant enzymes was done, namely by stopped-flow spectrophotometry. It is shown that H. pullorum SdHb reacts with NO much more quickly than TrHb, and that the three Prxs react promptly with peroxynitrite, Prx3 displaying the highest reactivity. Altogether, the results unveil SdHb and Prx3 as major protective systems of H. pullorum against nitrosative stress

Downregulation of the Drosophila Immune Response by Peptidoglycan-Recognition Proteins SC1 and SC2

Peptidoglycan-recognition proteins (PGRPs) are evolutionarily conserved molecules that are structurally related to bacterial amidases. Several Drosophila PGRPs have lost this enzymatic activity and serve as microbe sensors through peptidoglycan recognition. Other PGRP family members, such as Drosophila PGRP-SC1 or mammalian PGRP-L, have conserved the amidase function and are able to cleave peptidoglycan in vitro. However, the contribution of these amidase PGRPs to host defense in vivo has remained elusive so far. Using an RNA-interference approach, we addressed the function of two PGRPs with amidase activity in the Drosophila immune response. We observed that PGRP-SC1/2–depleted flies present a specific over-activation of the IMD (immune deficiency) signaling pathway after bacterial challenge. Our data suggest that these proteins act in the larval gut to prevent activation of this pathway following bacterial ingestion. We further show that a strict control of IMD-pathway activation is essential to prevent bacteria-induced developmental defects and larval death

Role of AmiA in the Morphological Transition of Helicobacter pylori and in Immune Escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection

Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor

Common Cell Shape Evolution of Two Nasopharyngeal Pathogens

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring.

BACKGROUND: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. METHODOLOGY/PRINCIPAL FINDING: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. CONCLUSIONS/SIGNIFICANCE: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies

NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM

BACKGROUND: Various Pseudomonas strains can use L-lactate as their sole carbon source for growth. However, the L-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. METHODOLOGY/PRINCIPAL FINDINGS: An NAD-independent L-lactate dehydrogenase (L-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of L-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), L-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on L-lactate, but retained the ability to grow on pyruvate. CONCLUSIONS/SIGNIFICANCE: It is proposed that L-iLDH plays an indispensable function in Pseudomonas L-lactate utilization by catalyzing the conversion of L-lactate into pyruvate

Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism

Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure–activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO4, R57A, and HpSK•shikimate-3-phosphate•ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A•162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism

Helicobacter pylori versus the Host: Remodeling of the Bacterial Outer Membrane Is Required for Survival in the Gastric Mucosa

Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4′-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4′-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4′-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4′-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host