Key Role of Staphylococcal Fibronectin-Binding Proteins During the Initial Stage of Staphylococcus aureus Keratitis in Humans

ObjectivesStaphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis.MethodsHuman corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells.ResultsSuperficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium (p < 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin (p < 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium (p < 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt.ConclusionS. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium.

The Effect of Benzyl Isothiocyanate On The Expression of Genes Encoding NADH Oxidase And Fibronectin-Binding Protein In Oral Streptococcal Biofilms

Background: Recent studies have shown that antibiotic treatment results in up- or down regulation of several virulence-associated genes. The genes encoding NADH oxidase (nox) and fibronectin-binding protein (fbp) are known to play important roles in biofilms of some oral bacterial species. The objective was to study the effect of benzyl isothiocyanate (BITC), an antimicrobial agent from Miswak plant, on the expression of nox and fbp genes in some oral streptococci.Methods: Bacterial strains were grown as biofilms in brucella broth. The crystal violet stained biofilms were quantified by optical density measurements at 590 nm. The biofilms were treated with an antimicrobial agent (BITC) for 2 h and fold change in mRNA expression of nox and fbp genes in BITC treated selected oral streptococci was measured by comparative ∆∆Ct method on Real-Time PCR machine.Results: The highest amount of biofilm mass was produced by A. defectiva, followed by S. gordonii, S. mutans, G. elegans and G. adiacens. Upon treatment with BITC, S. gordonii biofilms showed highest mRNA expression for both fbp and nox genes. Mean (SE) folds increase in the expression of nox mRNA: S. gordonii 2 (0.30), followed by S. mutans 1.25 (0.18), A. defectiva 1.03 (0.09), G. adiacens 0.7 (0.03). Similarly for fbp, folds increase in mRNA expression was: S. gordonii 2.65 (0.03), followed by A. defectiva 2.09 (0.60), G. elegans 1.61 (0.40), S. mutans 1.57 (0.20), and G. adiacens 0.58 (0.06). G. elegans mRNA levels for nox were extremely low (0.006-fold). Conclusion: BITC treatment of the biofilms caused an upregulation of biofilm-associated genes fbp and nox genes in most of the tested species suggesting the significance of these genes in biofilm lifestyle of these oral bacteria. Increased expression of nox and fbp genes in the biofilm lifestyle of these species needs further investigation to understand if it contributes to antimicrobial resistance.

Molecular Investigation of Fibronectin-Binding Protein Genes and Capacity of Biofilm Production in Staphylococcus Aureus Isolated From Clinical Samples

Biofilm production increases Staphylococcus aureus resistance to antibiotics and also host defense mechanisms. The current study aims to evaluate the biofilm formation by S. aureus and to determine the prevalence of fibronectin-binding protein genes, also its correlation with drug resistance. In this study, 100 clinical isolates of S. aureus were collected. The antibiotic susceptibility pattern of the isolates was evaluated by the disk agar diffusion method. The ability of biofilm formation in the studied isolates was also determined by microplate colorimetric assay. Then, all isolates were screened by polymerase chain reaction for the fnbA and fnbB genes. Out of 100 clinical isolates of S. aureus, the highest and lowest antibiotic resistance rates were against penicillin (94%) and vancomycin (6%). Thirty-two cases were found to be multi-drug resistant (MDR) among the all strains. The ability of biofilm production was observed in 89% of the isolates. The PCR results showed that the prevalence of fnbA and fnbB genes were 91% and 17%, respectively. Moreover, 100% and 21.8% of the MDR strains harbored the fnbA and fnbB genes respectively. The ability to form biofilm in MDR isolates of S. aureus is more than non-MDR isolates, especially fnbA positive ones. As the bacteria in the biofilm are difficult to kill by antibiotics, attention to the removal or control of the biofilm production seems to be necessary.

Bacterial carriage of genes encoding fibronectin-binding proteins is associated with long-term persistence of Staphylococcus aureus in the nasal and gut microbiota of infants

Staphylococcus aureus can colonize both the anterior nares and the gastrointestinal tract. However, colonization at these sites in the same individuals has not been studied, and the traits that facilitate colonization and persistence at these sites have not been compared. Samples from the nostrils and fecal collected on 9 occasions from 3 days to 3 years of age in 65 infants were cultured; 54 samples yielded S. aureus. The numbers of nasal and fecal S. aureus increased rapidly during the first weeks and were similar at 1 month of age (>40% of infants colonized). Thereafter, nasal carriage declined, while fecal carriage remained high during the first year of life. Individual strains were identified and their colonization patterns were related to their carriage of genes encoding adhesins and superantigenic toxins. Strains retrieved from both the nose and gut (n=44) of an infant were 4.5-times more likely to colonize long-term (≥3 weeks at both sites) than strains found only in the rectum/feces (n=56) or only in the nose (n=32) (P≤0.001). Gut colonization was significantly associated with carriage of the fnbA gene, and long-term colonization at either site was associated with carriage of fnbA and fnbB. In summary, gut colonization by S. aureus was more common than nasal carriage by S. aureus in the studied infants. Gut strains may provide a reservoir for invasive disease in vulnerable individuals. Fibronectin-binding adhesins and other virulence factors may facilitate commensal colonization and confer pathogenic potential. IMPORTANCE S. aureus may cause severe infections and frequently colonizes the nose. Nasal carriage of S. aureus increases three-fold the risk of invasive S. aureus infection. S. aureus is also commonly found in the gut microbiota of infants and young children. However, the relationships between the adhesins and other virulence factors of S. aureus strains and its abilities to colonize the nostrils and gut of infants are not well-understood. Our study explores the simultaneous colonization by S. aureus of the nasal and intestinal tracts of newborn infants, through 3 years of follow-up. We identify bacterial virulence traits that appear to facilitate persistent colonization of the nose and gut by S. aureus. This expands our current knowledge of the interplay between bacterial commensalism and pathogenicity. Moreover, it may contribute to the development of targeted strategies for combating S. aureus infection.

The Campylobacter jejuni CadF and FlpA fibronectin binding proteins facilitate signaling via host cell focal adhesions to inhibit cell motility and impede wound repair

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni infected cells. We found that C. jejuni infection of epithelial cells results in an increased focal adhesion size, enhanced signaling, and altered topology, as demonstrated by confocal microscopy, immunoblots, and super-resolution iPALM. Infection by C. jejuni also resulted in an increase in cell adhesion strength, reduced host cell motility, and reduction of collective host cell migration, a fundamental step in intestinal villi healing. Mechanistic studies demonstrated that the C. jejuni fibronectin-binding proteins CadF and FlpA are involved in the changes in focal adhesion dynamics and alterations in cell behavior. These findings are important because they provide a putative mechanistic basis for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacterial adhesins that target extracellular matrix components can alter cell behavior by manipulating focal adhesions.

Fisiopatología y factores de virulencia del streptococcus pyogenes implicados en la erisipela, celulitis y fascitis necrotizante

Sin tratamiento oportuno, las lesiones cutáneas por Streptococcus pueden poner en peligro la vida de una persona. En el caso de México hay factores para que se den estas infecciones por el incremento en el número de enfermos con diabetes mellitus tipo 2, que produce un estado de inmunosupresión en el organismo. Las vías de entrada para la bacteria pueden ser desde una tiña de los pies, un mal rasurado, hasta traumatismos graves. En el caso de S. pyogenes puede manifestarse desde una erisipela o una celulitis hasta una fascitis necrotizante o síndrome de shock tóxico estreptocóccico. Estas lesiones están relacionadas con los factores de virulencia de la bacteria que le permiten adherirse a la epidemis (fibronectin-binding, proteína M), evadir el sistema inmune (Mac-1, Mac-2, EndoS, SpeB, la cápsula, ScrpA) y comenzar un proceso de patogénesis (estreptolisina O, estreptolisina S, hialuronidasa, estreptocinasa, superantígenos). El objetivo de este trabajo es mostrar la fisiopatología de los principales factores de virulencia del Streptococo pyogenes que generan lesiones cutáneas.

The Increased Accumulation of Staphylococcus aureus Virulence Factors Is Maximized in a purR Mutant by the Increased Production of SarA and Decreased Production of Extracellular Proteases

ABSTRACT Mutation of purR was previously shown to enhance the virulence of Staphylococcus aureus in a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureus virulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role. Mutation of purR was also shown previously to result in increased abundance of SarA. Here, we demonstrate by transposon sequencing that mutation of purR in the USA300 strain LAC increases fitness in a biofilm while mutation of sarA has the opposite effect. Therefore, we assessed the impact of sarA on reported purR-associated phenotypes by characterizing isogenic purR, sarA, and sarA/purR mutants. The results confirmed that mutation of purR results in increased abundance of alpha toxin, protein A, the fibronectin-binding proteins, and SarA, decreased production of extracellular proteases, an increased capacity to form a biofilm, and increased virulence in an osteomyelitis model. Mutation of sarA had the opposite effects on all of these phenotypes and, other than bacterial burdens in the bone, all of the phenotypes of sarA/purR mutants were comparable to those of sarA mutants. Limiting the production of extracellular proteases reversed all of the phenotypes of sarA mutants and most of those of sarA/purR mutants. We conclude that a critical component defining the virulence of a purR mutant is the enhanced production of SarA, which limits protease production to an extent that promotes the accumulation of critical S. aureus virulence factors.