Non-fimbrial Adhesin Research Articles

Klebsiella Pneumoniae – Taxonomy, Occurrence, Identification, Virulence Factors and Pathogenicity

Abstract Gram-negative bacilli Klebsiella pneumoniae are among the most important pathogens responsible for healthcare-associated infections (HAIs). These bacteria often have high pathogenic and epidemic potential, contributing to infection outbreaks worldwide. K. pneumoniae is part of the natural microbiota of humans. At the same time, as an opportunistic microorganism, when the host organism is weakened, it can cause serious infections such as pneumonia, urinary tract infections, septic infections and intra-organ abscesses. Widespread distribution in nature and exceptional adaptability provide K. pneumoniae with the opportunity to master new niches in the hospital environment, which poses a threat to hospitalized patients. Also, the bacteria are increasingly causing life-threatening infections in the non-hospital environment. The pathogenicity of K. pneumoniae is determined by the presence of many virulence factors such as capsular polysaccharide (CPS, K antigen), lipopolysaccharid (LPS, O antigen), fimbrial and non-fimbrial adhesins, siderophores (aero-bactin, enterobactin, salmochelin and yersiniabactin), heat-stabile and heat-labile enterotoxins, cytotoxins and biofilm-forming ability. Currently, hypervirulent strains of K. pneumoniae (hvKp) equipped with new virulence traits constitute a significant danger. The paper presents these bacteria concerning the global threat arising from the dynamic spread of hvKp strains in hospitals in Poland and worldwide.

Infection by a multidrug-resistant Corynebacterium diphtheriae strain: prediction of virulence factors, CRISPR-Cas system analysis, and structural implications of mutations conferring rifampin resistance.

Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance.

The Trimeric Autotransporter Adhesin EmaA and Infective Endocarditis.

Infective endocarditis (IE), a disease of the endocardial surface of the heart, is usually of bacterial origin and disproportionally affects individuals with underlying structural heart disease. Although IE is typically associated with Gram-positive bacteria, a minority of cases are caused by a group of Gram-negative species referred to as the HACEK group. These species, classically associated with the oral cavity, consist of bacteria from the genera Haemophilus (excluding Haemophilus influenzae), Aggregatibacter, Cardiobacterium, Eikenella, and Kingella. Aggregatibacter actinomycetemcomitans, a bacterium of the Pasteurellaceae family, is classically associated with Aggressive Periodontitis and is also concomitant with the chronic form of the disease. Bacterial colonization of the oral cavity serves as a reservoir for infection at distal body sites via hematological spreading. A. actinomycetemcomitans adheres to and causes disease at multiple physiologic niches using a diverse array of bacterial cell surface structures, which include both fimbrial and nonfimbrial adhesins. The nonfimbrial adhesin EmaA (extracellular matrix binding protein adhesin A), which displays sequence heterogeneity dependent on the serotype of the bacterium, has been identified as a virulence determinant in the initiation of IE. In this chapter, we will discuss the known biochemical, molecular, and structural aspects of this protein, including its interactions with extracellular matrix components and how this multifunctional adhesin may contribute to the pathogenicity of A. actinomycetemcomitans.

Spermidine constitutes a key determinant of motility and attachment of Salmonella Typhimurium through a novel regulatory mechanism

Spermidine is a poly-cationic molecule belonging to the family of polyamines and is ubiquitously present in all organisms. Salmonella synthesizes, and harbours specialized transporters to import spermidine. A group of polyamines have been shown to assist in Salmonella Typhimurium's virulence and regulation of Salmonella pathogenicity Inslad 1 (SPI-1) genes and stress resistance; however, the mechanism remains elusive. The virulence trait of Salmonella depends on its ability to employ multiple surface structures to attach and adhere to the surface of the target cells before invasion and colonization of the host niche. Our study discovers the mechanism by which spermidine assists in the early stages of Salmonella pathogenesis. For the first time, we report that Salmonella Typhimurium regulates spermidine transport and biosynthesis processes in a mutually inclusive manner. Using a mouse model, we show that spermidine is critical for invasion into the murine Peyer’s patches, which further validated our in vitro cell line observation. We show that spermidine controls the mRNA expression of fimbrial (fimA) and non-fimbrial adhesins (siiE, pagN) in Salmonella and thereby assists in attachment to host cell surfaces. Spermidine also regulated the motility through the expression of flagellin genes by enhancing the translation of sigma-28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, spermidine tunes the expression of the two-component system BarA/SirA to regulate SPI-1 encoded genes. Thus, our study unravels a novel regulatory mechanism by which spermidine exerts critical functions during Salmonella Typhimurium pathogenesis.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Lactuca sativa (Lettuce).

Salmonella enterica serovar Typhimurium is a major cause of foodborne gastroenteritis. Recent outbreaks of infections by S. enterica serovar Typhimurium are often associated with non-animal-related food, i.e., vegetables, fruits, herbs, sprouts, and nuts. One main problem related to the consumption of fresh produce is the minimal processing, especially for leafy green salads. In this study, we focused on butterhead lettuce (Lactuca sativa) to which S. enterica serovar Typhimurium adheres at higher rates compared to Valerianella locusta, resulting in prolonged persistence. Here, we systematically analyzed factors contributing to adhesion of S. enterica serovar Typhimurium to L. sativa leaves. Application of a reductionist, synthetic approach, including the controlled surface expression of specific adhesive structures of S. enterica serovar Typhimurium, one at a time, enabled the identification of relevant fimbrial and nonfimbrial adhesins, the O-antigen of lipopolysaccharide, the flagella, and chemotaxis being involved in binding to L. sativa leaves. The analyses revealed contributions of Lpf fimbriae, Sti fimbriae, autotransported adhesin MisL, T1SS-secreted BapA, intact lipopolysaccharide (LPS), and flagella-mediated motility to adhesion of S. enterica serovar Typhimurium to L. sativa leaves. In addition, we identified BapA as a potential adhesin involved in binding to V. locusta and L. sativa leaf surfaces. IMPORTANCE The number of produce-associated outbreaks by gastrointestinal pathogens is increasing and underlines the relevance to human health. The mechanisms involved in the colonization of, persistence on, and transmission by, fresh produce are poorly understood. Here, we investigated the contribution of adhesive factors of S. enterica serovar Typhimurium in the initial phase of plant colonization, i.e., the binding to the plant surface. We used the previously established reductionist, synthetic approach to identify factors that contribute to the surface binding of S. enterica serovar Typhimurium to leaves of L. sativa by expressing all known adhesive structures by remote control expression system.

Molecular characteristics of fluoroquinolone-resistant Escherichia coli isolated from suckling piglets with colibacillosis

ObjectivesColibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates from suckling piglets with colibacillosis.ResultsA total of 43 FQ-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration (≥ 64 mg/L for ciprofloxacin, ≥ 128 mg/L for enrofloxacin, and ≥ 256 mg/L for norfloxacin). Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6’)-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%); β-lactamases–encoding blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%); and the chloramphenicol resistance genes (10 isolates, 83.3%); the sulfonamide resistance genes sul1 (9 isolates, 75.0%) and sul2 (10 isolates, 83.3%); the aminoglycoside modifying enzyme gene aac(3)-II (2 isolates, 16.7%). The F4 (7 isolates, 58.3%), LT:STb:EAST1 (5 isolates, 41.7%), and paa (3 isolates, 25.0%) were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively.ConclusionsBecause FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.

Into the understanding the multicellular lifestyle of Proteus mirabilis on solid surfaces.

Indwelling urinary catheterization can lead to the development of catheter-associated urinary tract infections (CAUTIs), an important type of nosocomial infection, as well as other medical issues among institutionalized adults. Recently, Proteus mirabilis was highlighted as the important cause of CAUTIs. The pathogenicity of P. mirabilis is dependent on two multicellular types of surface colonization: the adherence and swarming motility. Adhesion, mostly mediated by fimbrial and nonfimbrial adhesins, is important for the initiation of biofilm formation. Moreover, the production of urease frequently results in biofilm crystallization, which leads to the blockage of catheters. The heterologous polymeric matrix of the biofilm offers protection against antibiotics and the host immune system. P. mirabilis displays remarkable motility abilities. After contact with solid surfaces, hyper-flagellated cells are able to rapidly migrate. The importance of swarming motility in CAUTIs development remains controversial; however, it was indicated that swarming cells were able to co-express other virulence factors. Furthermore, flagella are strong immunomodulating proteins. On the other hand, both biofilm formation and swarming motility implicates multiple inter- and intraspecies interactions, which might contribute to the pathogenicity.

Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with periodontal disease and multiple disseminated extra-oral infections. Colonization of these distinct physiological niches is contingent on the expression of specific surface proteins during the initiation of developing biofilms. In this investigation, we studied fimbriae and three well-characterized nonfimbrial surface proteins (EmaA, Aae, and ApiA/Omp100) for their contribution to biofilm formation. Mutations of these proteins in multiple strains covering four different serotypes demonstrated variance in biofilm development that was strain dependent but independent of serotype. In a fimbriated background, only inactivation of emaA impacted biofilm mass. In contrast, inactivation of emaA and/or aae affected biofilm formation in nonfimbriated A. actinomycetemcomitans strains, whereas inactivation of apiA/omp100 had little effect on biofilm formation. When these genes were expressed individually in Escherichia coli, all transformed strains demonstrated an increase in biofilm mass compared to the parent strain. The strain expressing emaA generated the greatest mass of biofilm, whereas the strains expressing either aae or apiA/omp100 were greatly reduced and similar in mass. These data suggest a redundancy in function of these nonfimbrial adhesins, which is dependent on the genetic background of the strain investigated.

Nonfimbrial Adhesin Mutants Reveal Divergent Escherichia coli O157:H7 Adherence Mechanisms on Human and Cattle Epithelial Cells.

Shiga toxin-producing, enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a major foodborne pathogen causing symptoms ranging from simple intestinal discomfort to bloody diarrhea and life-threatening hemolytic uremic syndrome in humans. Cattle can be asymptomatically colonized by O157:H7 predominantly at the rectoanal junction (RAJ). Colonization of the RAJ is highly associated with the shedding of O157:H7 in bovine feces. Supershedding (SS) is a phenomenon that has been reported in some cattle that shed more than 104 colony-forming units of O57:H7 per gram of feces, 100–1000 times more or greater than normal shedders. The unique bovine RAJ cell adherence model revealed that O157:H7 employs a LEE-independent mechanism of attachment to one of the RAJ cell types, the squamous epithelial (RSE) cells. Nine nonfimbrial adhesins were selected to determine their role in the characteristic hyperadherent phenotype of SS O157 on bovine RSE cells, in comparison with human HEp-2 cells. A number of single nucleotide polymorphisms (SNPs) were found amongst these nonfimbrial adhesins across a number of SS isolates. In human cells, deletion of yfaL reduced the adherence of both EDL933 and SS17. However, deletion of eae resulted in a significant loss of adherence in SS17 whereas deletion of wzzB and iha in EDL933 resulted in the same loss of adherence to HEp-2 cells. On RSE cells, none of these nonfimbrial deletion mutants were able to alter the adherence phenotype of SS17. In EDL933, deletion of cah resulted in mitigated adherence. Surprisingly, four nonfimbrial adhesin gene deletions were actually able to confer the hyperadherent phenotype on RSE cells. Overall, this study reveals that the contribution of nonfimbrial adhesins to the adherence mechanisms and functions of O157:H7 is both strain and host cell type dependent as well as indicates a possible role of these nonfimbrial adhesins in the SS phenotype exhibited on RSE cells.

Prevalence and Characterization of Virulence Genes in <i>Escherichia coli</i> Isolated from Diarrheic Piglets in Korea

Enterotoxigenic Escherichia coli is one of the major causative infectious agents of diarrhea in newborn and post-weaning pigs and leads to a large economic loss worldwide. However, there is limited information on the distribution and characterization of virulence genes in E. coli isolated from diarrheic piglets, which also applies to the current status of pig farms in Korea. To investigate the prevalence and characterization of virulence genes in E. coli related to diarrhea in piglets, the rectal swab samples of diarrheic piglets (aged 2 d to 6 w) were collected from 163 farms between 2013 and 2016. Five to 10 individual swab samples from the same farm were pooled and cultured on MacConkey agar plates, and E. coli were identified using the API 32E system. Three sets of multiplex PCRs were used to detect 13 E. coli virulence genes. As a result, a total of 172 E. coli isolates encoding one or more of the virulence genes were identified. Among them, the prevalence of individual virulence gene was as follows, (1) fimbrial adhesins (43.0%): F4 (16.9%), F5 (4.1%), F6 (1.7%), F18 (21.5%), and F41 (3.5%); (2) toxins (90.1%): LT (19.2%), STa (20.9%), STb (25.6%), Stx2e (15.1%), EAST1 (48.3%); and (3) non-fimbrial adhesin (19.6%): EAE (14.0%), AIDA-1 (11.6%) and PAA (8.7%), respectively. Taken together, various pathotypes and virotypes of E. coli were identified in diarrheic piglets. These results suggest a broad array of virulence genes is associated with coliform diarrhea in piglets in Korea.

Recombinant protein production and purification of SiiD, SiiE and SiiF - Components of the SPI4-encoded type I secretion system from Salmonella Typhimurium

In humans, Salmonella enterica infections are responsible for a plethora of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secretes SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Secondary structure content and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure contents were in good agreement with estimates derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 Å resolution.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Corn Salad (Valerianella locusta).

Salmonella enterica is a foodborne pathogen often leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, frequency of outbreaks linked to the consumption of fresh or minimally processed food of nonanimal origin is increasing. New infection routes of S. enterica by vegetables, fruits, nuts, and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g., salads, that are mainly consumed in a minimally processed form. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species and other vegetables. In this study, we systematically analyzed the contributions of the complete adhesiome, of lipopolysaccharide (LPS), and of flagellum-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to Valerianella locusta (corn salad). We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad, with particular regard to the expression of all known adhesive structures, using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagellum-mediated motility of STM in adhesion to corn salad leaves.IMPORTANCE Transmission of gastrointestinal pathogens by contaminated fresh produce is of increasing relevance to human health. However, the mechanisms of contamination of, persistence on, and transmission by fresh produce are poorly understood. We investigated the contributions of the various adhesive structures of STM to the initial event in transmission, i.e., binding to the plant surface. A reductionist system was used that allowed experimentally controlled surface expression of individual adhesive structures and analyses of the contribution to binding to leave surfaces of corn salad under laboratory conditions. The model system allowed the determination of the relative contributions of fimbrial and nonfimbrial adhesins, the type 3 secretion systems, the O antigen of lipopolysaccharide, the flagella, and chemotaxis of STM to binding to corn salad leaves. Based on these data, future work could reveal the mechanism of binding and the relevance of interaction under agricultural conditions.

Comparative genomics of a subset of Adherent/Invasive Escherichia coli strains isolated from individuals without inflammatory bowel disease

There is increased evidence demonstrating the association between Crohn's Disease (CD), a type of Inflammatory Bowel Disease (IBD), and non-diarrheagenic Adherent/Invasive Escherichia coli (AIEC) isolates. AIEC strains are phenotypically characterized by their adhesion, invasion and intra-macrophage survival capabilities. In the present study, the genomes of five AIEC strains isolated from individuals without IBD (four from healthy donors and one from peritoneal liquid) were sequenced and compared with AIEC prototype strains (LF82 and NRG857c), and with extra-intestinal uropathogenic strain (UPEC CFT073). Non-IBD-AIEC strains showed an Average Nucleotide Identity up to 98% compared with control strains. Blast identities of the five non-IBD-AIEC strains were higher when compared to AIEC and UPEC reference strains than with another E. coli pathotypes, suggesting a relationship between them. The SNPs phylogeny grouped the five non-IBD-AIEC strains in one separated cluster, which indicates the emergence of these strains apart from the AIEC group. Additionally, four genomic islands not previously reported in AIEC strains were identified. An incomplete Type VI secretion system was found in non-IBD-AIEC strains; however, the Type II secretion system was complete. Several groups of genes reported in AIEC strains were searched in the five non-IBD-AIEC strains, and the presence of fimA, fliC, fuhD, chuA, irp2 and cvaC were confirmed. Other virulence factors were detected in non-IBD-AIEC strains, which were absent in AIEC reference strains, including EhaG, non-fimbrial adhesin 1, PapG, F17D-G, YehA/D, FeuC, IucD, CbtA, VgrG-1, Cnf1 and HlyE. Based on the differences in virulence determinants and SNPs, it is plausible to suggest that non-IBD AIEC strains belong to a different pathotype.

Virulence and antimicrobial resistance profiles of Escherichia coli encoding mcr gene from diarrhoeic weaned piglets in Korea during 2007-2016.

This study aimed to analyse the presence of mcr genes and virulence genes in Escherichia coli (E. coli) isolates from diarrhoeic piglets between 2007-2016 in Korea. A total of 364 E. coli isolates were obtained from diarrhoeic weaned piglets between 2007-2016. Minimum inhibitory concentrationss were determined according to the Clinical and Laboratory Standards Institute (CLSI). DNA samples were tested for the presence of mcr-1, mcr-2 and mcr-3 genes. Furthermore, multiplex PCR was used to detect toxin, fimbrial and non-fimbrial adhesin genes. It was found that 2.5% (nine of 364) of the isolates carried the mcr genes. Four isolates carried the mcr-1 gene and eight isolates had the mcr-3 gene. Three isolates carried both mcr-1 and mcr-3 genes; the mcr-2 gene was not found. All isolates carrying mcr genes were multidrug-resistant. F18 (77.8%, seven of nine) and Stx2e (44.4%, four of nine) were frequently detected in E. coli carrying the mcr gene. In conclusion, it would appear that the most prevalent mcr gene of E. coli from diarrhoeic weaned piglets in Korea was mcr-3. It is believed that this is the first report of two plasmid-mediated colistin resistance genes - mcr-1 and mcr-3 - coexisting in the same isolates (0258, 0491, 0516) from piglets with diarrhoea in Korea. Those mcr-positive isolates showed multidrug resistance and the majority of those encoded Stx2e and F18. This indicates the risk of inefficient treatment for oedema disease in weaned piglets.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to β-lactam and non-β-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Active sulfite oxidase domain of Salmonella enterica pathogenic protein small intestine invasive factor E (SiiE): a potential diagnostic target.

Serovars of Salmonella enterica are common food-borne bacterial pathogens. Salmonella typhi, which causes typhoid, is the most dangerous of them. Though detailed molecular pathogenesis studies reveal many virulence factors, inability to identify their biochemical functions hampers the development of diagnostic methods and therapeutic leads. Lack of quicker diagnosis is an impediment in starting early antibiotic treatment to reduce the severe morbidity and mortality in typhoid. In this study, employing bioinformatic prediction, biochemical analysis, and recombinantly cloning the active region, we show that extracellularly secreted virulence-associated protein, small intestinal invasion factor E (SiiE), possesses a sulfite oxidase (SO) domain that catalyzes the conversion of sodium sulfite to sodium sulfate using tungsten as the cofactor. This activity common to Salmonella enterica serovars seems to be specific to them from bioinformatic analysis of available bacterial genomes. Along with the ability of this large non-fimbrial adhesin of 600kDa binding to sialic acid on the host cells, this activity could aid in subverting the host defense mechanism by destroying sulfites released by the immune cells and colonize the host gastrointestinal epithelium. Being an extracellular enzyme, it could be an ideal candidate for developing diagnostics of S. enterica, particularly S. typhi.

TosR-Mediated Regulation of Adhesins and Biofilm Formation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli strains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (type one secretion A) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. The tosRCBDAEF operon encodes the secretory machinery necessary for TosA localization to the E.coli cell surface, as well as the transcriptional regulator TosR. TosR binds upstream of the tos operon and in a concentration-dependent manner either induces or represses tosA expression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of the pap operon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on the E.coli CFT073 genome, we induced expression of tosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation.IMPORTANCE Uropathogenic E.coli strains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.

Distribution of Major Pilin Subunit Genes Among Atypical Enteropathogenic Escherichia coli and Influence of Growth Media on Expression of the ecp Operon.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coli pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94–100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1–8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coli common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37°C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

Prevalence of pathogenicity island ETT2 in Escherichia coli isolated from piglets with diarrhea in northeast of China.

Our aim was to investigate the prevalence of the pathogenicity island ETT2 and to examine the relationship between the ETT2 locus and other virulence factors in Escherichia coli (E. coli) isolated from piglets with diarrhea. A total of 354 E. coli strains isolated from scouring piglets were tested using PCR for the presence of the ETT2 locus. The E. coli strains were also analyzed for enterotoxins, fimbriae, non-fimbrial adhesin, Shiga toxins, pathogenicity islands, α-haemolysin (hlyA), afa8 gene cluster and autotransporter protease (sepA) genes. The results showed that 215 (60.7%) of the isolates possessed the ETT2 island. In 215 ETT2-positive E. coli strains, the virulence genes found were EAST1 (27.0%), irp2 (18.6%), paa (15.4%), STb (7.9%), LT (6.5%), ler (4.7%), hlyA (3.7%), AIDA-I (3.7%), K88 (3.7%), eae (3.3%), STa (2.8%), afaD (1.4%), afaE (1.4%), K99 (0.9%) and sepA (0.47%), respectively, and the isolates could be assigned into 25 different virulence factor patterns. In 139 ETT2-negative E. coli strains, the virulence genes detected were EAST1 (38.9%), paa (14.4%), STb (11.5%), AIDA-I (10.1%), irp2 (7.9%), sepA (2.16%), LT (0.7%), STa (0.7%), eae (0.7%), ler (0.7%), hlyA (0.7%) and K88 (0.7%), respectively, and the isolates could be classified into 13 different virulence factor patterns. Moreover, the occurrence of LT gene of ETT2-positive E. coli strains was far more than that of ETT2-negative E. coli strains.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica.

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.