Host Cell Adhesion Research Articles

Development of a LAMP assay for rapid detection of different intimin variants of attaching and effacing microbial pathogens

Intimin harboured by pathogenic Escherichia coli (E. coli) strains is a key virulence factor involved in host cell adherence and colonization. Twenty-seven intimin-encoding E. coli attaching and effacing (eae) gene variants have been reported according to their 3' binding domain sequences. In our study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect all known intimin variants. Four primers specific for six regions of eae genes were designed using online software. The eae-LAMP assay was highly specific and detected all 27 tested eae variants; no cross-reactions were observed with genes from enterotoxigenic E. coli (ETEC), E. coli BL21, Salmonella, Shigella, Listeria monocytogenes, or Streptococcus suis type 2 (SS2). With the lowest detection limit of approximately 10 copies per reaction the eae-LAMP assay was 100 times more sensitive than conventional PCR. These results, and the results of tests involving food and faecal samples artificially contaminated with E. coli O157 : H7 (eaeγ+), show that the eae-LAMP assay is a simple, rapid, sensitive and specific tool for detecting intimin variants from pathogenic strains of E. coli. The eae-LAMP assay has great potential for wider applications, not only in the laboratory but also in the field setting, as it does not require specialized equipment.

Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection

BackgroundViral upper respiratory tract infections are associated with increased colonization by Streptococcus pneumoniae but the mechanisms underlying this relationship are unclear. The objective of this study is to describe a comprehensive picture of the cellular interaction between the adhering bacteria and host cells in the presence or absence of a viral co-infection.ResultsGene expression profiles of Detroit-562 pharyngeal cells, which were either mock-infected or infected with human respiratory syncytial virus (RSV) or human parainfluenza virus 3 (HPIV3), were analyzed using human microarrays. Transcription response of S. pneumoniae strain TIGR4 (serotype 4) in the presence of either mock- or viral-infected cells was analyzed by pneumococcal microarray. Significantly regulated genes were identified by both significance analysis of microarray (SAM) and a ≥ 2-fold change ratio cut-off. The adherence of S. pneumoniae to human pharyngeal cells was significantly augmented in the presence of RSV or HPIV3 infection. Global gene expression profiling of the host cells during infection with RSV or HPIV3 revealed increased transcription of carcinoembryonic antigen-related cell adhesion molecules (CEACAM1), CD47, fibronectin, interferon-stimulated genes and many other host cell adhesion molecules. Pneumococci increased transcription of several genes involved in adhesive functions (psaA, pilus islet), choline uptake and incorporation (lic operon), as well as transport and binding.ConclusionsWe have identified a core transcriptome that represents the basic machinery required for adherence of pneumococci to D562 cells infected or not infected with a virus. These bacterial genes and cell adhesion molecules can potentially be used to control pneumococcal adherence occurring secondary to a viral infection.

Immunoglobulin domains inEscherichia coliand other enterobacteria: from pathogenesis to applications in antibody technologies

The immunoglobulin (Ig) protein domain is widespread in nature having a well-recognized role in proteins of the immune system. In this review, we describe the proteins containing Ig-like domains in Escherichia coli and enterobacteria, reporting their structural and functional properties, protein folding, and diverse biological roles. In addition, we cover the expression of heterologous Ig domains in E. coli owing to its biotechnological application for expression and selection of antibody fragments and full-length IgG molecules. Ig-like domains in E. coli and enterobacteria are frequently found in cell surface proteins and fimbrial organelles playing important functions during host cell adhesion and invasion of pathogenic strains, being structural components of pilus and nonpilus fimbrial systems and members of the intimin/invasin family of outer membrane (OM) adhesins. Ig-like domains are also found in periplasmic chaperones and OM usher proteins assembling fimbriae, in oxidoreductases and hydrolytic enzymes, ATP-binding cassette transporters, sugar-binding and metal-resistance proteins. The folding of most E. coli Ig-like domains is assisted by periplasmic chaperones, peptidyl-prolyl cis/trans isomerases and disulfide bond catalysts that also participate in the folding of antibodies expressed in this bacterium. The technologies for expression and selection of recombinant antibodies in E. coli are described along with their biotechnological potential.

Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells

Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.

Multi-functional analysis of Klebsiella pneumoniae fimbrial types in adherence and biofilm formation

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with nosocomially acquired infections. Host cell adherence and biofilm formation of K. pneumoniae isolates is mediated by type 1 (T1P) and type 3 (MR/K) pili whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. The E. coli common pilus (ECP) is an adhesive structure produced by all E. coli pathogroups and a homolog of the ecpABCDE operon is present in the K. pneumoniae genome. In this study, we aimed to determine the prevalence of these three fimbrial genes among a collection of 69 clinical and environmental K. pneumoniae strains and to establish a correlation with fimbrial production during cell adherence and biofilm formation. The PCR-based survey demonstrated that 96% of the K. pneumoniae strains contained ecpA and 94% of these strains produced ECP during adhesion to cultured epithelial cells. Eighty percent of the strains forming biofilms on glass produced ECP, suggesting that ECP is required, at least in vitro, for expression of these phenotypes. The fim operon was found in 100% of the strains and T1P was detected in 96% of these strains. While all the strains examined contained mrkA, only 57% of them produced MR/K fimbriae, alone or together with ECP. In summary, this study highlights the ability of K. pneumoniae strains to produce ECP, which may represent a new important adhesive structure of this organism. Further, it defines the multi-fimbrial nature of the interaction of this nosocomial pathogen with host epithelial cells and inert surfaces.

Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg

BackgroundBovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection.ResultsThe results show, however, that none of these soluble forms are able to block viral infectivity. To better understand the role of gB during BoHV-4 lytic replication, a recombinant BoHV-4 was generated by homologous recombination from a BoHV-4 cloned as a Bacterial artificial chromosome (BAC) (pBAC-BoHV-4-A), in which most of the BoHV-4 gB ORF was substituted by the insertion of a DNA stuffer selectable cassette. The resulting recombinant BoHV-4 genome (pBAC-BoHV-4-AΔgB-KanaGalK) was completely unable to reconstitute infectious replicating viral particles (Infectious Replicating Viral Particles, IRVPs) and to replicate when transfected in permissive cell lines in comparison to its revertant clone (pBAC-BoHV-4-ΔgB-Rev) or pBAC-BoHV-4-A parental clone.ConclusionThis demonstrates that the BoHV-4 replicating cycle is dependent on gB. Moreover, when gB was deleted from a recombinant BoHV-4 genome delivering an heterologous glycoprotein, Vesicular Stomatitis Virus Glycoprotein (VSVg), VSVg was unable to complement gB. This study provides direct evidence that gB is necessary for BoHV-4 lytic replication.

Overexpression of luxS Cannot Increase Autoinducer‐2 Production, Only Affect the Growth and Biofilm Formation in Streptococcus suis

LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules via luxS-based autoinducer-2 (AI-2). LuxS has been reported to plays critical roles in regulating various behaviors of bacteria. AI-2 is a byproduct of the catabolism of S-adenosylhomocysteine (SAH) performed by the LuxS and Pfs enzymes. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as an inability to produce AI-2 were observed in bacteria having a luxS mutant gene. In this study, the level of AI-2 activity exhibits a growth-phase dependence with a maximum in late exponential culture in SS. An SS strain that overexpressed luxS was constructed to comprehensively understand the function of AI-2. Overexpressed luxS was not able to increase the level of pfs expression and produce additional AI-2, and the bacteria were slower growing and produced only slightly more biofilm than the wild type. Thus, AI-2 production is not correlated with luxS transcription. luxS expression is constitutive, but the transcription of pfs is perhaps correlated with AI-2 production in SS.

An improved quantitative method to assess adhesive properties of Trichomonas vaginalis to host vaginal ectocervical cells using flow cytometry

Microbial adhesion is a critical step for infection and colonization of the host. Trichomonas vaginalis, a human urogenital extracellular parasite, relies on host cell adhesion for infection and pathogenesis. Although host cell adhesion of T. vaginalis is strain-dependent and it may be influenced by many environmental factors, a technical limitation to quantify T. vaginalis adhesion falls upon a laborious and time-consuming protocol of fluorescent microscopy. This technical limitation reduces the ability of screening multiple parameters or detecting multiple cell types simultaneously. Here we tested the capability of using flow cytometry as a qualitative and quantitative method to measure adhesion of this human infectious microorganism to vaginal ectocervical cells. Various strains of T. vaginalis with different adhesion properties were stained with CellTracker Orange (CMTMR) prior to incubation with host cells. Analyses by flow cytometry revealed that adhered CMTMR-stained parasites were clearly distinguishable from the host cells and also enabled absolute cell counts to be determined. This method was validated with the comparison of parasite strains that display variable degrees of host cell adhesion. This assay can now be applied to test many variables and environmental factors simultaneously that may affect T. vaginalis adhesion.

Structural basis for promiscuity and specificity during Candida glabrata invasion of host epithelia

The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization.

Oxidative stress resistance genes contribute to the pathogenic potential of the anaerobic protozoan parasite, Entamoeba histolytica

The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite’s adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization.

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes – survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%–0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10tm1Cgn mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.

Cigarette Smoke Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress

The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biofilm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biofilm dispersal, and increased transcription of biofilm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biofilms. Exposure of bacteria to hydrogen peroxide alone increased biofilm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biofilm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding fibronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized fibronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans.

Candida Species: New Insights into Biofilm Formation

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.

Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo

Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

The E. coli CsgB nucleator of curli assembles to β-sheet oligomers that alter the CsgA fibrillization mechanism

Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli, Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower and appears to proceed through large amorphous aggregates before forming filaments. Substoichiometric concentrations of CsgB induce a change in the mechanism of CsgA aggregation from that of forming amorphous aggregates to that of structured intermediates similar to those of CsgB alone. Oligomeric CsgB accelerated the aggregation of CsgA, in contrast to monomeric CsgB, which had no effect. The structured β-strand oligomers formed by CsgB serve as nucleators for CsgA aggregation. These results provide insights into the formation of curli in vivo, especially the nucleator function of CsgB.

Comparative genomics of bacteria in the genus Providencia isolated from wild Drosophila melanogaster

BackgroundComparative genomics can be an initial step in finding the genetic basis for phenotypic differences among bacterial strains and species. Bacteria belonging to the genus Providencia have been isolated from numerous and varied environments. We sequenced, annotated and compared draft genomes of P. rettgeri, P. sneebia, P. alcalifaciens, and P. burhodogranariea. These bacterial species that were all originally isolated as infections of wild Drosophila melanogaster and have been previously shown to vary in virulence to experimentally infected flies.ResultsWe found that these Providencia species share a large core genome, but also possess distinct sets of genes that are unique to each isolate. We compared the genomes of these isolates to draft genomes of four Providencia isolated from the human gut and found that the core genome size does not substantially change upon inclusion of the human isolates. We found many adhesion related genes among those genes that were unique to each genome. We also found that each isolate has at least one type 3 secretion system (T3SS), a known virulence factor, though not all identified T3SS belong to the same family nor are they in syntenic genomic locations.ConclusionsThe Providencia species examined here are characterized by high degree of genomic similarity which will likely extend to other species and isolates within this genus. The presence of T3SS islands in all of the genomes reveal that their presence is not sufficient to indicate virulence towards D. melanogaster, since some of the T3SS-bearing isolates are known to cause little mortality. The variation in adhesion genes and the presence of T3SSs indicates that host cell adhesion is likely an important aspect of Providencia virulence.

Investigating the Surface Structure and Antibody Recognition Forces of Tannerella Forsythia by Scanning Probe Microscopy

Tannerella forsythia ATCC 43037 (Tf) is a Gram-negative oral anaerobe which is one of the major contributors to periodontal disease in humans [1]. It possesses a glycosylated S-layer consisting of two regularly arrayed subunits as outermost cell envelope layer. The S-layer of Tf was shown to be a virulence factor, capable of delaying the bacterium's recognition by the innate immune system [2] and mediating adhesion and invasion of host cells [3]. In this study, we used scanning probe microscopy to probe the S-layer surface of live Tf cells by investigating the nano subunit structure so as to characterize the surface structure and the interactions of Tf S-layer proteins. High-resolution imaging of living Tf wild-type cells has shown a periodic square lattice structure with about 10 nm dimension, while on the surface of an S-layer single mutant no periodic structure was visible. In addition, single-molecule force spectroscopy using a TfsA S-layer protein specific antibody immobilized onto AFM tips revealed specific interaction forces with the S-layer structure on living Tf wild-type cells. Our data contribute to the elucidation of a surface structure on living bacteria and to the understanding of their subunit arrangement with nano-meter resolution.[1] S. Holt, and J. Ebersole Periodontology 2000 38 (2000) 72.[2] G. Sekot, G. Posch, P. Messner, M. Matejka, X. Rausch-Fan, O. Andrukhov, and C. Schaffer, J. Dent, Res 90 (2011) 109[3] J. Sakakibara, K. Nagano, and Y. Murakami, Microbiology 153 (2007) 866.

Involvement of T6 Pili in Biofilm Formation by Serotype M6 Streptococcus pyogenes

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.

Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus

BackgroundStaphylococcus aureus is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by fnbA/B, are major pathogenesis determinants in these infections through their involvement in S. aureus adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics, frequently occurring in vivo because of impaired drug diffusion at the infection site, can alter S. aureus phenotype. We therefore investigated their impact on S. aureus fibronectin-mediated adhesiveness and invasiveness.MethodsAfter in vitro challenge of S. aureus 8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored fnbA/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts.ResultsOxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in fnbA/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting fnbA/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or fnbA/B transcription levels. Only oxacillin-treated S. aureus displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls.ConclusionOur findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in S. aureus in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled in vitro conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during in vivo infections is thus still uncertain and requires further investigations.