Amino‐terminal residues dictate the export efficiency of the Campylobacter jejuni filament proteins via the flagellum

Bacterial flagella play an essential role in the pathogenesis of numerous enteric pathogens. The flagellum is required for motility, colonization, and in some instances, for the secretion of effector proteins. In contrast to the intensively studied flagella of Escherichia coli and Salmonella typhimurium, the flagella of Campylobacter jejuni, Helicobacter pylori and Vibrio cholerae are less well characterized and composed of multiple flagellin subunits. This study was performed to gain a better understanding of flagellin export from the flagellar type III secretion apparatus of C. jejuni. The flagellar filament of C. jejuni is comprised of two flagellins termed FlaA and FlaB. We demonstrate that the amino‐termini of FlaA and FlaB determine the length of the flagellum and motility of C. jejuni. We also demonstrate that protein‐specific residues in the amino‐terminus of FlaA and FlaB dictate export efficiency from the flagellar type III secretion system (T3SS) of Yersinia enterocolitica. These findings demonstrate that key residues within the amino‐termini of two nearly identical proteins influence protein export efficiency, and that the mechanism governing the efficiency of protein export is conserved among two pathogens belonging to distinct bacterial classes. These findings are of additional interest because C. jejuni utilizes the flagellum to export virulence proteins.     

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA supercoiling.     

Correlation Between In Vitro Secretion of Virulence-Associated Proteins of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and Colonization of Chickens

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture–conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.     

The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics.     

Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens

The Dsb family of redox proteins catalyzes disulfide bond formation and isomerization. Since mutations indsb genes change the conformation and stability of many extracytoplasmic proteins, and since many virulence factors of pathogenic bacteria are extracytoplasmic, inactivation ofdsb genes often results in pathogen attenuation. This study investigated the role of 2 membrane-bound oxidoreductases, DsbB and DsbI, in theCampylobacter jejuni oxidative Dsb pathway.Campylobacter mutants, lacking DsbB or DsbI or both, were constructed by allelic replacement and used in the human intestinal epithelial T84 cell line for the gentamicin protection assay (invasion assay) and chicken colonization experiments. InC. coli strain 23/1, the inactivation of thedsbB ordsbI gene separately did not significantly affect the colonization process. However, simultaneous disruption of both membrane-bound oxidoreductase genes significantly decreased the strain’s ability to colonize chicken intestines. Moreover,C. jejuni strain 81–176 with mutateddsbB ordsbI genes showed reduced invasion/intracellular survival abilities. No cells of the double mutants (dsbB ⁻ dsbI ⁻) ofC. jejuni 81–176 were recovered from human cells after 3 h of invasion.     

Structure of a sigma28-regulated nonflagellar virulence protein from Campylobacter jejuni

Campylobacter jejuni, a Gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. Although the mechanism of C.jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. Cj0977 is a recently identified virulence factor in C. jejuni and is expressed by a σ²⁸ promoter that controls late genes in the flagellar regulon. A Cj0977 mutant strain is fully motile but significantly reduced in the invasion of intestinal epithelial cells in vitro. Here, we report the crystal structure of the major structural domain of Cj0977, which reveals a homodimeric “hot-dog” fold architecture. Of note, the characteristic hot-dog fold has been found in various coenzyme A (CoA) compound binding proteins with numerous oligomeric states. Structural comparison with other known hot-dog fold proteins locates a putative binding site for an acyl-CoA compound in the Cj0977 protein. Structure-based site-directed mutagenesis followed by invasion assays indicates that key residues in the putative binding site are indeed essential for the Cj0977 virulence function, suggesting a possible function of Cj0977 as an acyl-CoA binding regulatory protein.     

Pathogenicity ofCampylobacter jejuni in intraperitoneally or intravenously inoculated ferrets

Ferret kits inoculated intravenously (IV) withCampylobacter jejuni after pretreatment with parenteral iron developed more severe systemic signs and more prolonged bacteremia than untreated inoculated controls. Watery diarrhea began in both groups 2–16 h after inoculation and lasted less than 48 h.C. jejuni was cultured from rectal swabs 2–8 h after inoculation, and gut colonization persisted up to 15 days, suggesting that colonization does not necessarily induce diarrhea. Gut colonization occurred as rapidly after IV inoculation of ferrets in which the common bile duct had been ligated as it did in unligated controls.C. jejuni apparently reached the intestinal lumen by mucosal invasion from the bloodstream. Bacteremia following natural infection could thus result in repeated passages ofC. jejuni across the gut wall, exposing the mucosa to both the bacterial cells and their metabolic products. Histological evidence of an inflammatory response in the mucosa, without severe epithelial damage, suggests a toxin-mediated secretory diarrhea.     

Cell surface‐associated aggregation‐promoting factor from Lactobacillus gasseri SBT2055 facilitates host colonization and competitive exclusion of Campylobacter jejuni

Campylobacter jejuni, one of the most common causes of gastroenteritis worldwide, is transmitted to humans through poultry. We previously reported that Lactobacillus gasseri SBT2055 (LG2055) reduced C. jejuni infection in human epithelial cells in vitro and inhibited pathogen colonization of chickens in vivo. This suggested that the LG2055 adhesion and/or co‐aggregation phenotype mediated by cell‐surface aggregation‐promoting factors (APFs) may be important for the competitive exclusion of C. jejuni. Here, we show that cell surface‐associated APF1 promoted LG2055 self‐aggregation and adhesion to human epithelial cells and exhibited high affinity for the extracellular matrix component fibronectin. These effects were absent in the apf1 knockout mutant, indicating the role of APF1 in LG2055‐mediated inhibition of C. jejuni in epithelial cells and chicken colonization. Similar to APF1, APF2 promoted the co‐aggregation of LG2055 and C. jejuni but did not inhibit C. jejuni infection. Our data suggest a pivotal role for APF1 in mediating the interaction of LG2055 with human intestinal cells and in inhibiting C. jejuni colonization of the gastrointestinal tract. We thus provide new insight into the health‐promoting effects of probiotics and mechanisms of competitive exclusion in poultry. Further research is needed to determine whether the probiotic strains reach the epithelial surface.     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter ( yplA ). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242 , and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram‐negative pathogen Salmonella typhimurium and the Gram‐positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.     

Campylobacter jejuni survival within human epithelial cells is enhanced by the secreted protein CiaI

Although it is known that Campylobacter jejuni invade the cells that line the human intestinal tract, the bacterial proteins that enable this pathogen to survive within Campylobacter-containing vacuoles (CCV) have not been identified. Here, we describe the identification and characterization of a protein that we termed CiaI for Campylobacter invasion antigen involved in intracellular survival. We show that CiaI harbours an amino-terminal type III secretion sequence and is secreted from C. jejuni through the flagellar type III secretion system. In addition, the ciaI mutant was impaired in intracellular survival when compared with a wild-type strain, as judged by the gentamicin-protection assay. Fluorescence microscopy examination of epithelial cells infected with the C. jejuni ciaI mutant revealed that the CCV were more frequently co-localized with Cathepsin D (a lysosomal marker) than the CCV in cells infected with a C. jejuni wild-type strain. Ectopic expression of CiaI-GFP in epithelial cells yielded a punctate phenotype not observed with the other C. jejuni genes, and this phenotype was abolished by mutation of a dileucine motif located in the carboxy-terminus of the protein. Based on the data, we conclude that CiaI contributes to the ability of C. jejuni to survive within epithelial cells.     

不同宿主源弯曲菌黏附侵袭肠上皮细胞及在鸡肠道内定殖的比较

【背景】弯曲菌(Campylobacter)是重要的人畜共患病原菌,可在多种动物肠道定殖,但不同宿主源弯曲菌对肠上皮细胞的黏附侵袭特征及在鸡肠道内的定殖能力并不明确。【目的】探究不同宿主源弯曲菌对不同宿主肠上皮细胞黏附侵袭及在鸡肠道内定殖能力的差异性。【方法】利用 5株来自不同宿主源弯曲菌,包括人源、鸡源、鸭源和牛源空肠弯曲菌(Campylobacter jejuni)及猪源结肠弯曲菌(Campylobacter coli),在对菌株PCR鉴定、运动力及生物膜形成能力测定的基础上,分别测定各菌株对人源肠上皮细胞Caco-2、猪源肠上皮细胞IPEC-J2和大鼠源肠上皮细胞IEC-6的黏附能力,通过庆大霉素保护试验测定菌株对肠上皮细胞的侵袭能力,比较黏附量和侵袭量的差异;将5株弯曲菌分别口服攻毒鸡,于攻毒后不同日龄(different days post inoculation,DPI)采集肠道样品测定弯曲菌的菌落数,比较不同弯曲菌在鸡肠道内定殖的差异。【结果】人源弯曲菌运动力显著高于其他4株动物源弯曲菌,而牛源和猪源弯曲菌生物膜形成能力显著高于其他菌株。黏附侵袭测定结果显示,人源弯曲菌对Caco-2细胞的黏附能力显著高于动物源弯曲菌,但侵袭能力显著低于动物源弯曲菌;鸭源和牛源弯曲菌对IPEC-J2细胞的黏附能力显著低于其他菌株,而且鸭源弯曲菌的侵袭能力显著低于其他菌株;不同菌株对IEC-6细胞的黏附能力无显著差异,但鸡源弯曲菌侵袭能力显著低于其他菌株。不同弯曲菌口服攻毒鸡后1、3和6d动物源弯曲菌定殖水平显著高于人源,在攻毒后10d和15d仅牛源弯曲菌显著高于人源,于攻毒后15d所有菌株达到约8-10Log₁₀(CFU/g)的稳定定殖水平。【结论】来源于不同宿主的弯曲菌对不同宿主肠上皮细胞均具有黏附侵袭能力,同时可在鸡肠道内稳定定殖,提示弯曲菌在不同动物间传播和适应性定殖的特征,对开展弯曲菌针对性防控措施具有一定的借鉴意义。     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.