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Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plf(QT598), wherein plfG was replaced by papG; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. IMPORTANCE Fimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin-encoding plfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. A plf mutant also showed decreased colonization of the kidneys in a mouse competitive infection model. PL fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.

Objective This report describes the case of a patient with chronic radial nerve entrapment symptoms managed with chiropractic care. We propose a complementary functional neurologic assessment of muscle function in different positions that could reveal muscle dysfunctions absent with standard test position. Clinical Features A 45-year-old man presented to a private chiropractic clinic with a throbbing pain 5 cm above the right lateral elbow epicondyle radiating onto the back of the lower arm and increasing after using a mouse when working on a computer. A Mill test and a Cozen test created pain near the lateral epicondylitis. The use of complementary functional neurologic assessment for radial nerve entrapment showed changes in manual muscle testing after tests were done in different positions to increase the compression on the nerve. Intervention and Outcome Chiropractic management was performed, including myofascial therapy, spinal and proximal radioulnar joint adjustments, neural mobilization, and the use of a splint. After 7 days (2 treatments), the patient showed no elbow pain even if he worked on his computer using a mouse. After a 2-year follow-up, no recurrence was reported. Conclusion In this case of radial nerve entrapment symptoms, the patient benefited from chiropractic management using standard chiropractic, applied kinesiology, and neural mobilization techniques. The complementary functional neurologic assessment of radial nerve entrapment proposed revealed muscles dysfunctions absent with the standard test position. These changes in manual muscle testing were useful to determine the possible sites of entrapment in order to direct the therapeutic efforts to these locations.

Fimbrial adhesins play a critical role for bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified a fimbrial gene cluster belonging to the π group that we named PL (P-like) fimbriae, since genetic organization and sequence are similar to Pap and related fimbriae. Screening of genomic databases indicated that genes encoding PL fimbriae located on IncF plasmids are present in a diversity of E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit sequence divergence in adhesin encoding genes, and strains could be divided into 5 classes based on differences in sequences of the PlfG adhesin protein. The plf genes from two predominant PlfG adhesin classes, PlfG-I and PlfG-II were cloned. PL fimbriae were visualized by electron microscopy, promoted biofilm formation, demonstrated distinct hemagglutination profiles and promoted adherence to human bladder and kidney epithelial cell lines. Hybrid fimbriae comprised of genes from plfQT598 wherein plfG was replaced by papG encoding adhesin genes were also shown to be functional and mediate adherence to epithelial cells, further indicating similarity and functional compatibility between these two types of fimbriae. Although deletion of plf genes did not significantly reduce colonization of the mouse urinary tract, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences and similarities to P fimbriae and may contribute to adherence to cells and colonization of host tissues.ImportanceFimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin encoding PlfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. Such fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.

Urinary tract infections (UTIs) are a common bacterial infectious disease in humans, and strains of uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTIs. During infection, UPEC must cope with a variety of stressful conditions in the urinary tract. Here, we demonstrate that the small RNA (sRNA) RyfA of UPEC strains is required for resistance to oxidative and osmotic stresses. Transcriptomic analysis of the ryfA mutant showed changes in expression of genes associated with general stress responses, metabolism, biofilm formation and genes coding for cell surface proteins. Inactivation of ryfA in UPEC strain CFT073 decreased urinary tract colonization in mice and the ryfA mutant also had reduced production of type 1 and P fimbriae (pili), adhesins which are known to be important for UTI. Furthermore, loss of ryfA also reduced UPEC survival in human macrophages. Thus, ryfA plays a key regulatory role in UPEC adaptation to stress, which contributes to UTI and survival in macrophages., Author summary Small regulatory RNAs play a critical role in bacterial adaptation, physiology, and metabolism. We investigated the role of the small RNA ryfA in uropathogenic E. coli, by using genetic, molecular and cell culture approaches, and mouse infection models. We demon-strate that RyfA is important for resistance to oxidative and osmotic stresses and affects the global transcription of multiple responses to stress. Deletion of ryfA also reduced production of type 1 and P fimbriae (pili) and increased swimming motility. RyfA also plays an important role in urinary tract infection (UTI) in the mouse model and for survival in human primary macrophages and cell lines. Elucidating a role for RyfA in adaptation to stress response and virulence provides insight into new regulatory mechanisms important for virulence in pathogenic E. coli and likely other Enterobacteria that may lead to development of targeted therapeutic or prophylactic approaches against these pathogens.

Plus de vingt ans apres la definition du concept de mediation culturelle, propose par Elisabeth Caillet et Evelyne Lehalle, les quatre codirecteurs de La Mediation culturelle : cinquieme roue du carrosse ? presentent un ouvrage ancre tant dans la pratique que dans la theorie. Fanny Serain et Patrick Chazottes, en preface du recueil qu’ils codirigent avec Francois Vaysse et Elisabeth Caillet, disent vouloir modestement « repositionner l’avancement de certains reperes, dans une profession en co...

TagB, TagC (tandem autotransporter genes B and C), and Sha (Serine-protease hemagglutinin autotransporter) are recently described members of the SPATE (serine protease autotransporters of Enterobacteriaceae) family. These SPATEs can cause cytopathic effects on bladder cells and contribute to urinary tract infection in a mouse model. Bladder epithelial cells form an important barrier in the urinary tract. Some SPATEs produced by pathogenic E. coli are known to breach the bladder epithelium. The capacity of these newly described SPATEs to alter bladder epithelial cells and the role of the serine protease active site were investigated. All three SPATE proteins were internalized by bladder epithelial cells and altered the distribution of actin cytoskeleton. Sha and TagC were also shown to degrade mucin and gelatin respectively. Inactivation of the serine catalytic site in each of these SPATEs did not affect secretion of the SPATEs from bacterial cells, but abrogated entry into epithelial cells, cytotoxicity, and proteolytic activity. Thus, our results show that the serine catalytic triad of these proteins is required for internalization in host cells, actin disruption, and degradation of host substrates such as mucin and gelatin.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for various infections outside the gastrointestinal tract in humans and other animals. ExPEC strain MT78 is invasive to various nonphagocytic cells and highly virulent in vivo To identify genes required for invasion of nonphagocytic cells by this strain, we applied signature-tagged mutagenesis to generate a library of mutants and tested them for invasion of avian fibroblasts. Mutants showing reduced cellular invasion included those with insertions in the fim operon, encoding type 1 fimbriae. Another attenuated mutant showed a disruption in the treA gene, which encodes a periplasmic trehalase. The substrate of TreA, trehalose, can be metabolized and used as a carbon source or can serve as an osmoprotectant under conditions of osmotic stress in E. coli K-12. We generated and characterized mutant MT78ΔtreA In contrast to the wild type, MT78ΔtreA was able to grow under osmotic stress caused by 0.6 M urea but not in minimal M9 medium with trehalose as the only carbon source. It presented decreased association and invasion of avian fibroblasts, decreased yeast agglutination titer, and impaired type 1 fimbria production. In a murine model of urinary tract infection, MT78ΔtreA was less able to colonize the bladder. All phenotypes were rescued in the complemented mutant. Our results show that the treA gene is needed for optimal production of type 1 fimbriae in ExPEC strain MT78 and that loss of treA significantly reduces its cell invasion capacity and colonization of the bladder in a murine model of urinary tract infection.

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC . This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

Avian pathogenic Escherichia coli (APEC) causes respiratory disease and sepsis in poultry. To persist in its host, E. coli requires essential nutrients including iron. Since iron is limited in extra-intestinal tissues, E. coli produces siderophores, small molecules with high affinity for ferric iron, to sequester this essential nutrient. To counter bacterial siderophore systems, mammalian hosts secrete siderocalin (also called lipocalin 2 or NGAL), which binds ferric-siderophore complexes rendering them unavailable to bacteria. In humans and mice, siderocalin is known to play a role in primary defense against bacterial infections. In poultry, 4 proteins display homology to the human NGAL (CALβ, CALγ, Ggal-C8GC and Ex-FABP). The function and expression of the genes coding for these 4 proteins during infection by APEC is still unknown. Expression levels of these genes were determined by quantitative RT-PCR using RNA extracted from lungs, livers and spleens of healthy 3-week-old chickens and chickens infected with APEC. The gene coding for Ex-FABP was overexpressed in all organs tested. It was significantly more overexpressed in the lungs and liver than in the spleen (37.3 and 27.3 times versus 11.5 times, respectively). The genes coding for Calβ and Calγ were also found significantly overexpressed in the liver (27 and 8.2 times, respectively). To confirm the function of Ex-FABP as a siderocalin, the gene coding for this protein was cloned in an expression vector and the protein was purified. In vitro growth inhibition of E. coli strains by Ex-FABP was assayed in parallel with growth inhibition caused by human siderocalin. Purified Ex-FABP inhibited growth of E. coli K-12, which only produces the siderophore enterobactin. However, E. coli strains producing pathogen-associated siderophores including salmochelins (glucosylated enterobactin), aerobactin and yersiniabactin grew normally in the presence of Ex-FABP. These results indicate that Ex-FABP is an avian siderocalin with a siderophore-binding activity similar to that of human siderocalin and that pathogen-specific siderophores are required by APEC to overcome this innate defense protein in poultry.

International audience; The genotoxin colibactin synthesized by Escherichia coli is a secondary metabolite belonging to the chemical family of hybrid polyketide/non-ribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the High-Pathogenicity Island, encoding the siderophore yersiniabactin that belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90 involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to the eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence

International audience; Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

Objectives This report describes the case of a patient with chronic idiopathic meralgia paresthetica associated with bilateral sacroiliac joint dysfunction who was managed with chiropractic care. Clinical Features A 35-year-old white woman presented to a private chiropractic clinic with a complaint of numbness in the right anterolateral thigh region. Neurological assessment revealed a diminution of sensibility and discrimination on the right lateral femoral cutaneous nerve territory. Pain was rated as 8.5 on a numeric pain scale of 0 to 10. Musculoskeletal examination of the pelvic region disclosed bilateral sacroiliac joint dysfunction. Intervention and Outcomes Chiropractic management included pelvic mobilizations, myofascial therapy, transverse friction massage, and stretching exercises. After 3 visits (2 weeks later), result of neurological evaluation was normal, with no residual numbness over the lateral thigh. Conclusion In the present case, chiropractic management with standard and applied kinesiology techniques resulted in recovery of meralgia paresthetica symptoms for this patient.

In animals, somatic stimulation of the limbs can evoke sympathetic reflexes of supraspinal origin. In addition, spinal reflexes can be elicited by stimulation of somatic tissues of the trunk. However, limited evidence is available concerning the specific modulation of sympathetic reflexes by afferents from the thoracic spine. This has also been largely overlooked in healthy humans. The aim of the present study was to determine whether tonic noxious heat (NH) applied to the skin over T3-T5 could segmentally increase supraspinal sympathetic reflexes (skin conductance responses - SCRs) induced by phasic electrical stimulation of the sural nerve. In addition, the effect of spinal manipulation (SM) on SCR amplitude and SCR amplification by NH was investigated. During the control session, palmar and plantar SCR amplitude was stable, showing no significant modulation. During NH and SM, however, palmar SCR amplitude was respectively increased and decreased in comparison to baseline, leading to a robust difference in SCR amplitude between the 2 conditions (p

Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

This article describes the chiropractic clinical management and therapeutic benefits accruing to a patient with temporomandibular joint (TMJ) disorder and spinal muscular atrophy type III.A 35-year-old white man presented at the university chiropractic outpatient clinic with a complaint of masseter muscle pain and mouth-opening restriction. Temporomandibular joint range of motion evaluation revealed restricted opening (11 mm interincisival), and pain was rated by the patient at an intensity of 5 on a pain scale of 0 to 10.Chiropractic care was provided and included TMJ mobilization, myofascial therapy, trigger point therapy, and light spinal mobilizations of the upper cervical vertebrae. Final evaluation of TMJ range of motion showed active opening of 12 mm with absence of pain and muscle tenderness of the jaw.This case suggests that a patient with musculoskeletal disorders related to underlying neurodegenerative pathologies may benefit from chiropractic management adapted to their condition. In the present case, chiropractic treatment of the TMJ represented a viable, low-cost approach with limited adverse effects compared with surgery.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.

Introduction: Extraintestinal pathogenic Escherichia coli (ExPEC) is associated with various diseases such as urinary tract infections, neonatal meningitis and septicemia. There are many virulence factors (VF) encoded by genes in ExPEC, including papC, papG, ecpA, iroN, fyuA, iutA, ompTp, tsh, hlyF, hlyA and iss. These virulence genes may be present in pathogenicity islands (PAI) or plasmids. Methodology: In this study, we analyzed the presence of VF encoding genes, PAI sequences and phylogenetic groups of 96 ExPEC strains isolated from the urine and blood of patients at the University Hospital of Londrina, and we compared them with 50 faecal commensal strains from healthy individuals. Results: The VF fyuA (65.60%) was detected in pathogenic strains and commensal strains (46%). A comparison of the distribution of ExPEC and commensal strains in the phylogenetic groups showed that more ExPEC strains belonged to group B2 whereas more of the commensal isolates belonged to group A. The distribution of the seven PAI sequences between commensal strains and ExPEC strains showed that PAI IV536 was common in both ExPEC and commensal isolates. Conclusions: These results showed that the ExPEC strains that belonged to group B2 had more PAI sequences compared to those of the other groups, especially group B1, which had virulence genes but the lowest percentage of PAI sequences, which leads us to conclude that the virulence of ExPEC strains characterized as B2 is likely attributed to PAI encoded genes, whereas the virulence of ExPEC strains belonging to phylogenetic group B1 is likely due to plasmid encoded virulence genes.

Avian pathogenic Escherichia coli (APEC) strains harbor a number of virulence genes and cause extraintestinal diseases, such as septicemia, swollen-head syndrome, salpingitis, and omphalitis in poultry. APEC strains are not known to cause intestinal diseases. Herein, for the first time, it is reported that APEC strains were able to induce an enterotoxigenic-like effect in rabbit ligated ileal loops. Strain SEPT362 caused cell detachment of the intestinal villi, which also showed a flattened and wilted appearance, but the integrity of the tight junctions was maintained. Additionally, this strain did not adhere to enterocytes in vivo, although adhesin encoding genes (fimH, csgA, lpfA2-3, and ECP) were present while other lpfA types, sfa, afa, papC, and ral genes were not. This enterotoxigenic-like activity was conserved after thermal treatment of the supernatant at 65°C but not at 100°C. Moreover, experiments based on filtering with different molecular weight cut-off (MWCO) pore sizes demonstrated that the component associated with the observed biological effect has a molecular weight >100 kDa. Blast search and polymerase chain reaction assays for known E. coli virulence factors showed that strain SEPT362 harbors the gene encoding for the toxin EAST-1 and the serine protease autotransporter (SPATE) Tsh, but is negative for genes encoding for the toxins LT-I, STh, STp, Stx1, Stx2, CNF-1, CNF-2, CDT and the SPATEs Sat, Pic, Vat, SigA, SepA, EatA, EspP, or EspC. A cloned copy of the tsh gene in E. coli K-12 was also tested and was shown to have an enterotoxic effect. These results suggest that APEC might induce fluid accumulation in the rabbit gut. The Tsh autotransporter seems to be one of the factors associated with this phenotype. © Copyright 2014, Mary Ann Liebert, Inc. 2014.

Autotransporters are a large family of virulence factors of Gram-negative bacterial pathogens. The autotransporter adhesin involved in diffuse adherence (AIDA-I) is an outer membrane protein of Escherichia coli , which allows binding to epithelial cells as well as the autoaggregation of bacteria. AIDA-I is glycosylated by a specific heptosyltransferase encoded by the aah gene that forms an operon with the aidA gene. aidA is highly prevalent in strains that cause disease in pigs. Nevertheless, there are only two published whole-length sequences for this gene. In this study, we sequenced the aah and aidA genes of 24 aidA -positive porcine strains harboring distinct virulence factor profiles. We compared the obtained sequences and performed phylogenetic and pulsed-field electrophoresis analyses. Our results suggest that there are at least 3 different alleles for aidA , which are associated with distinct virulence factor profiles. The genes are found on high-molecular-weight plasmids and seem to evolve via shuffling mechanisms, with one of the sequences showing evidence of genetic recombination. Our work suggests that genetic plasticity allows the evolution of aah-aidA alleles that are selected during pathogenesis.

The pstSCAB-phoU operon encodes the phosphate-specific transport system (Pst). Loss of Pst constitutively activates the Pho regulon and decreases bacterial virulence. However, specific mechanisms underlying decreased bacterial virulence through inactivation of Pst are poorly understood. In uropathogenic Escherichia coli (UPEC) strain CFT073, inactivation of pst decreased urinary tract colonization in CBA/J mice. The pst mutant was deficient in production of type 1 fimbriae and showed decreased expression of the fimA structural gene which correlated with differential expression of the fimB , fimE , ipuA , and ipbA genes, encoding recombinases, mediating inversion of the fim promoter. The role of fim downregulation in attenuation of the pst mutant was confirmed using a fim phase-locked-on derivative, which demonstrated a significant gain in virulence. In addition, the pst mutant was less able to invade human bladder epithelial cells. Since type 1 fimbriae contribute to UPEC virulence by promoting colonization and invasion of bladder cells, the reduced bladder colonization by the pst mutant is predominantly attributed to downregulation of these fimbriae. Elucidation of mechanisms mediating the control of type 1 fimbriae through activation of the Pho regulon in UPEC may open new avenues for therapeutics or prophylactics against urinary tract infections.

Extra-intestinal pathogenic E. coli cause urinary tract infections (UTIs), newborn meningitis, abdominal sepsis and septicemia. UTIs affect millions of women annually, and result in significant health care costs and morbidity worldwide. Uropathogenic E. coli (UPEC) is the predominant urinary tract pathogen, causing up to 85% of UTIs. Despite appropriate therapy, recurrent episode of UTI are common and bacterial strains are increasingly more resistant to many currently used antimicrobial agents. The pstSCAB-phoU operon encodes the phosphate specific transport system (Pst) and belongs to the Pho regulon, which is regulated by the two-component regulatory system (TCRS) PhoBR. Inactivation of the Pst system in E. coli and other bacteria leads to constitutive activation of the Pho regulon, perturbations in cellular adaptation, and decreased virulence. The role of the Pst system in uropathogenic E. coli (UPEC) was assessed by deleting the pstSCA genes in UPEC strain CFT073. In competitive (co-challenge) and single-strain infections, the pst mutant was attenuated for colonization of both the bladder and kidneys of CBA/J mice and was impaired for production of Type 1 fimbriae. Type 1 fimbriae are essential for UPEC virulence and their phase-variable expression is positively and negatively regulated by FimB and FimE, respectively. In vitro, in LB broth and human urine, repression of the fim structural gene fimA in the pst mutant correlated with increased orientation of the fim promoter in the OFF-position. In vivo, down-regulation of fimA in CFT073 Δpst correlated with the up-regulation of fimE. To confirm the specific role of repression of fim expression by the pst mutant during UTI, fim phase locked-ON pst derivatives of the pst mutant and WT CFT073 strains were constructed. Compared to the pst mutant, the fim phase locked-ON pst derivative demonstrated a significant gain in colonization of the bladder, that was similar to that of CFT073 WT and CFT073 fim locked-ON strains. As Type 1 fimbriae are important for UPEC virulence, by promoting adhesion, our results suggest that the reduced bladder colonization by the pst mutant during UTI is predominantly attributed to down-regulation of these fimbriae. Since the Pho regulon is controlled by the TCRS PhoBR, molecules inducing the expression of the Pho regulon through inactivation of Pst or activation of PhoBR could potentially impair UPEC virulence by inhibiting colonization and the infection cycle, which is dependent on expression of type 1 fimbrial adhesins.

Roles of the ZnuACB and ZupT transporters were assessed in Escherichia coli K-12 and uropathogenic E. coli (UPEC) CFT073. K-12 and CFT073 Δ znu Δ zupT mutants demonstrated decreased 65 Zn 2+ uptake and growth in minimal medium. CFT073Δ znu demonstrated an intermediate decrease of 65 Zn 2+ uptake and growth in minimal medium, whereas the CFT073Δ zupT mutant grew as well as CFT073 and exhibited a less marked decrease in 65 Zn 2+ uptake. CFT073 mutants grew as well as the wild type in human urine. In competitive infections in CBA/J mice, the Δ zupT mutant demonstrated no disadvantage during urinary tract infection. In contrast, the UPEC Δ znu and Δ znu Δ zupT strains demonstrated significantly reduced numbers in the bladders (mean 4.4- and 30-fold reductions, respectively) and kidneys (mean 41- and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the Δ znu and Δ znu Δ zupT mutants were reduced in the kidneys ( P = 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 Δ znu Δ zupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.

Salmonella serovars contain a wide variety of putative fimbrial systems that may contribute to colonization of specific niches. Salmonella enterica serovar Typhi is the etiologic agent of typhoid fever and is a pathogen specific to humans. In a previous study, we identified a gene, STY3920 ( stgC ), encoding the predicted usher of the stg fimbrial operon, that was expressed by serovar Typhi during infection of human macrophages. The stg genes are located in the glmS - pstS intergenic region in serovar Typhi and certain Escherichia coli strains, but they are absent in other S. enterica serovars. We cloned the stg fimbrial operon into a nonfimbriate E. coli K-12 strain and into S. enterica serovar Typhimurium. We demonstrated that the stg fimbrial operon contributed to increased adherence to human epithelial cells. Transcriptional fusion assays with serovar Typhi suggested that stg is preferentially expressed in minimal medium. Deletion of stg reduced adherence of serovar Typhi to epithelial cells. However, deletion of stg increased uptake of serovar Typhi by human macrophages, and overexpression of stg in serovar Typhi and serovar Typhimurium strains reduced phagocytosis by human macrophages. These strains survived inside macrophages as well as the wild-type parent. Although the stgC gene contains a premature stop codon that disrupts the expected open reading frame encoding the usher and is therefore considered a pseudogene, our results show that the stg operon may encode a functional fimbria. Thus, this serovar Typhi-specific fimbrial operon contributes to interactions with host cells, and further characterization is important for understanding the role of the stg fimbrial cluster in typhoid fever pathogenesis.

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

In a previous study, ecs-3 , a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain χ7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg , is a member of a distinct group of related fimbriae that are located in the glmS - pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain χ7122 and an isogenic Δ stg mutant demonstrated that compared to the wild-type parent, the Δ stg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.