Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

The Campylobacter jejuni helical to coccoid transition involves changes to peptidoglycan and the ability to elicit an immune response.

Campylobacter jejuni is a prevalent enteric pathogen that changes morphology from helical to coccoid under unfavorable conditions. Bacterial peptidoglycan maintains cell shape. As C. jejuni transformed from helical to coccoid, peptidoglycan dipeptides increased and tri- and tetrapeptides decreased. The DL-carboxypeptidase Pgp1 important for C. jejuni helical morphology and putative N-acetylmuramoyl-L-alanyl amidase AmiA were both involved in the coccoid transition. Mutants in pgp1 and amiA showed reduced coccoid formation, with ∆pgp1∆amiA producing minimal coccoids. Both ∆amiA and ∆amiA∆pgp1 lacked flagella and formed unseparated chains of cells consistent with a role for AmiA in cell separation. All strains accumulated peptidoglycan dipeptides over time, but only strains capable of becoming coccoid displayed tripeptide changes. C. jejuni helical shape and corresponding peptidoglycan structure are important for pathogenesis-related attributes. Concomitantly, changing to a coccoid morphology resulted in differences in pathogenic properties; coccoid C. jejuni were non-motile and non-infectious, with minimal adherence and invasion of epithelial cells and an inability to stimulate IL-8. Coccoid peptidoglycan exhibited reduced activation of innate immune receptors Nod1 and Nod2 versus helical peptidoglycan. C. jejuni also transitioned to coccoid within epithelial cells, so the inability of the immune system to detect coccoid C. jejuni may be significant in its pathogenesis.

Morphology heterogeneity within a Campylobacter jejuni helical population: the use of calcofluor white to generate rod-shaped C. jejuni 81-176 clones and the genetic determinants responsible for differences in morphology within 11168 strains.

Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81-176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation. Whole genome sequencing of CFW-passaged strains showed no specific changes correlating to CFW exposure. The cjj81176_1279 (recR; recombinational DNA repair) and cjj81176_1449 (unknown function) genes were highly variable in all 81-176 strains sequenced. A frameshift mutation in pgp1 of our laboratory isolate of the straight genome sequenced variant of 11168 (11168-GS) was also identified. The PG muropeptide profile of 11168-GS was identical to that of Δpgp1 in the original minimally passaged 11168 strain (11168-O). Introduction of wild type pgp1 into 11168-GS did not restore helical morphology. The recR gene was also highly variable in 11168 strains. Microbial cell-to-cell heterogeneity is proposed as a mechanism of ensuring bacterial survival in sub-optimal conditions. In certain environments, changes in C. jejuni morphology due to genetic heterogeneity may promote C. jejuni survival.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4.

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

The Helical Shape of Campylobacter jejuni Promotes In Vivo Pathogenesis by Aiding Transit through Intestinal Mucus and Colonization of Crypts.

Campylobacter jejuni is a helix-shaped enteric bacterial pathogen and a common cause of gastroenteritis. We recently developed a mouse model for this human pathogen utilizing the SIGIRR-deficient mouse strain, which exhibits significant intestinal inflammation in response to intestinal C. jejuni infection. In the current study, this mouse model was used to define whether C. jejuni's characteristic helical shape plays a role in its ability to colonize and elicit inflammation in the mouse intestine. Mice were infected with the previously characterized straight-rod Δpgp1 and Δpgp2 mutant strains, along with a newly characterized curved-rod Δ1228 mutant strain. We also compared the resultant infections and pathology to those elicited by the helix-shaped wild-type C. jejuni and complemented strains. Despite displaying wild-type colonization of the intestinal lumen, the straight-rod Δpgp1 and Δpgp2 mutants were essentially nonpathogenic, while all strains with a curved or helical shape retained their expected virulence. Furthermore, analysis of C. jejuni localization within the ceca of infected mice determined that the primary difference between the rod-shaped, nonpathogenic mutants and the helix-shaped, pathogenic strains was the ability to colonize intestinal crypts. Rod-shaped mutants appeared unable to colonize intestinal crypts due to an inability to pass through the intestinal mucus layer to directly contact the epithelium. Together, these results support a critical role for C. jejuni's helical morphology in enabling it to traverse and colonize the mucus-filled intestinal crypts of their host, a necessary step required to trigger intestinal inflammation in response to C. jejuni.

The Campylobacter jejuni†CprRS two-component regulatory system regulates aspects of the cell envelope.

Campylobacter jejuni is a leading cause of food-borne gastroenteritis in humans. It lives commensally in the gastrointestinal tract of animals, and tolerates variable conditions during transit/colonization of susceptible hosts. The C. jejuni CprRS two-component system contains an essential response regulator (CprR), and deletion of the cprS sensor kinase enhances biofilms. We sought to identify CprRS-regulated genes and better understand how the system affects survival. Expression from the cprR promoter was highest during logarithmic growth and dependent on CprS. CprR(D52A) did not support viability, indicating that CprR phosphorylation is essential despite the dispensability of CprS. We identified a GTAAAC consensus bound by the CprR C-terminus; the Asp52 residue of full-length CprR was required for binding, suggesting phosphorylation is required. Transcripts differing in expression in ΔcprS compared with wildtype (WT) contained a putative CprR binding site upstream of their promoter region and encoded htrA (periplasmic protease upstream of cprRS) and peb4 (SurA-like chaperone). Consistent with direct regulation, the CprR consensus in the htrA promoter was bound by CprR(CTD). Finally, ΔhtrA formed enhanced biofilms, and ΔcprS biofilms were suppressed by Mg(2+). CprRS is the first C. jejuni regulatory system shown to control genes related to the cell envelope, the first line of interaction between pathogen and changing environments.

A novel mouse model of Campylobacter jejuni gastroenteritis reveals key pro-inflammatory and tissue protective roles for Toll-like receptor signaling during infection.

Campylobacter jejuni is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how C. jejuni colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, C. jejuni typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal C. jejuni colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (Sigirr(-/-)), a negative regulator of MyD88-dependent signaling led to heavy and widespread C. jejuni colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. C. jejuni heavily colonized the cecal and colonic crypts of Sigirr(-/-) mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established C. jejuni pathogenicity factors, capsular polysaccharides (kpsM) and motility/flagella (flaA). We also explored the basis for the inflammatory response elicited by C. jejuni in Sigirr(-/-) mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by C. jejuni. Despite heavy colonization, Tlr4(-/-)/Sigirr(-/-) mice were largely unresponsive to infection by C. jejuni, whereas Tlr2(-/-)/Sigirr(-/-) mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the C. jejuni capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of Sigirr(-/-) mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to C. jejuni.

Peptidoglycan LD-carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the DL-carboxypeptidase Pgp1.

Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

Peptidoglycan-modifying enzyme Pgp1 is required for helical cell shape and pathogenicity traits in Campylobacter jejuni.

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain - which similarly produced straight or kinked cells - exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster under iron limitation.

IMPORTANCE: Campylobacter jejuni is a bacterium that is prevalent in the ceca of farmed poultry such as chickens. Consumption of ill-prepared poultry is thus the most common route by which C. jejuni infects the human gut to cause a typically self-limiting but severe gastrointestinal illness that can be fatal to very young, old, or immunocompromised people. The lack of a vaccine and an increasing resistance to current antibiotics highlight a need to better understand the mechanisms that make C. jejuni a successful human pathogen. This study focused on the functional components of one such mechanism-a molecular system that helps C. jejuni thrive despite the restriction on growth-available iron by the human body, which typically defends against pathogens. In providing a deeper understanding of how this system functions, this study contributes toward the goal of reducing the enormous global socioeconomic burden caused by C. jejuni.

Multiple Campylobacter jejuni proteins affecting the peptidoglycan structure and the degree of helical cell curvature.

Campylobacter jejuni is a Gram-negative helical bacterium. Its helical morphology, maintained by the peptidoglycan (PG) layer, plays a key role in its transmission in the environment, colonization, and pathogenic properties. The previously characterized PG hydrolases Pgp1 and Pgp2 are important for generating C. jejuni helical morphology, with deletion mutants being rod-shaped and showing alterations in their PG muropeptide profiles in comparison to the wild type. Homology searches and bioinformatics were used to identify additional gene products involved in C. jejuni morphogenesis: the putative bactofilin 1104 and the M23 peptidase domain-containing proteins 0166, 1105, and 1228. Deletions in the corresponding genes resulted in varying curved rod morphologies with changes in their PG muropeptide profiles. All changes in the mutants complemented except 1104. Overexpression of 1104 and 1105 also resulted in changes in the morphology and in the muropeptide profiles, suggesting that the dose of these two gene products influences these characteristics. The related helical ε-Proteobacterium Helicobacter pylori has characterized homologs of C. jejuni 1104, 1105, and 1228 proteins, yet deletion of the homologous genes in H. pylori had differing effects on H. pylori PG muropeptide profiles and/or morphology compared to the C. jejuni deletion mutants. It is therefore apparent that even related organisms with similar morphologies and homologous proteins can have diverse PG biosynthetic pathways, highlighting the importance of studying PG biosynthesis in related organisms.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster in iron scavenging.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be antecedent to highly debilitating long-term sequelae. Expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution by each of the downstream genes ( fetABCDEF ) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA , fetB, fetC , and fetD , suggesting a role in iron acquisition for each encoded protein. FetA expression was modulated by iron-availability but not dependent on the presence of FetB, FetC, FetD, FetE or FetF. Functions of the putative thioredoxins FetE and FetF were redundant in iron scavenging, requiring a double deletion (Δ fetEF ) to exhibit a growth defect. C. jejuni FetE was expressed and the structure solved to 1.50 Å, revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than E. coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo . This study advances our understanding of the contributions by the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge towards mitigating C. jejuni -related morbidity and mortality.

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity.

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.

Hyperosmotic stress response of Campylobacter jejuni.

The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter(-1). C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.

FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection.

Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ΔfdhU ΔfdhA double mutant were similar to those of a ΔfdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ΔfdhU and ΔfdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.

5'-methylthioadenosine nucleosidase is implicated in playing a key role in a modified futalosine pathway for menaquinone biosynthesis in Campylobacter jejuni.

Menaquinone (vitamin K(2)) serves as an electron carrier in the electron transport chain required for respiration in many pathogenic bacteria. Most bacteria utilize a common menaquinone biosynthetic pathway as exemplified by Escherichia coli. Recently, a novel biosynthetic pathway, the futalosine pathway, was discovered in Streptomyces. Bioinformatic analysis strongly suggests that this pathway is also operative in the human pathogens Campylobacter jejuni and Helicobacter pylori. Here, we provide compelling evidence that a modified futalosine pathway is operative in C. jejuni and that it utilizes 6-amino-6-deoxyfutalosine instead of futalosine. A key step in the Streptomyces pathway involves a nucleosidase called futalosine hydrolase. The closest homolog in C. jejuni has been annotated as a 5'-methylthioadenosine nucleosidase (MTAN). We have shown that this C. jejuni enzyme has MTAN activity but negligible futalosine hydrolase activity. However, the C. jejuni MTAN is able to hydrolyze 6-amino-6-deoxyfutalosine at a rate comparable with that of its known substrates. This suggests that the adenine-containing version of futalosine is the true biosynthetic intermediate in this organism. To demonstrate this in vivo, we constructed a C. jejuni mutant strain deleted for mqnA2, which is predicted to encode for the enzyme required to synthesize 6-amino-6-deoxyfutalosine. Growth of this mutant was readily rescued by the addition of 6-amino-6-deoxyfutalosine, but not futalosine. This provides the first direct evidence that a modified futalosine pathway is operative in C. jejuni. It also highlights the tremendous versatility of the C. jejuni MTAN, which plays key roles in S-adenosylmethionine recycling, the biosynthesis of autoinducer molecules, and the biosynthesis of menaquinone.

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles.

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis.

Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed DeltagalT and DeltacstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (DeltawaaF and DeltalgtF but not DeltagalT or DeltacstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (DeltacstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The DeltawaaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization.

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Delta cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Delta cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Delta cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.