Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

Tropical pyomyositis: an update.

Tropical pyomyositis (TP) is a life-threatening bacterial infection of the skeletal muscle that occurs particularly among children, young adults and those with immunocompromised conditions. The appropriate diagnosis and treatment are often delayed due to its non-specific signs, leading to fatal consequences. Staphylococcus aureus, especially methicillin-susceptible S. aureus, is responsible for most TP cases. However, other bacteria (i.e. streptococci, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., Candida spp., Mycobacterium spp.) have been reported. This narrative review provides an update on the epidemiology and clinical course of TP. A special focus is laid on the role of toxins (i.e. Panton-Valentine Leucocidin and α-toxin) in the pathogenesis of TP and their implication for the clinical management of infection.

La pyomyosite tropicale (TP) est une infection bactérienne potentiellement mortelle du muscle squelettique qui survient particulièrement chez les enfants, les jeunes adultes et les personnes immunodéprimées. Le diagnostic et le traitement appropriés sont souvent retardés en raison de ses signes non spécifiques, entraînant des conséquences fatales. Staphylococcus aureus, en particulier S. aureus sensible à la méthicilline, est responsable de la plupart des cas de TP. Cependant, d'autres bactéries (ex: streptocoques, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., Candida spp., Mycobacterium spp.) ont été rapportées. Cette revue narrative fournit une mise à jour sur l'épidémiologie et l'évolution clinique du TP. Un accent particulier est mis sur le rôle des toxines (la Leukocidine de Panton-Valentine et l'α-toxine) dans la pathogenèse du TP et leur implication pour la prise en charge clinique de l'infection.

Superantigen SpeA attenuates the biofilm forming capacity of Streptococcus pyogenes.

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA) Requires Death Receptor Fas, in Addition to LFA-1, To Trigger Cell Death in T Lymphocytes.

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitansA. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent.

A two-amino acid mutation in murine IgA enables downstream processing and purification on staphylococcal superantigen-like protein 7.

With few exceptions, all currently marketed antibody therapeutics are IgG molecules. One of the reasons that other antibody isotypes are less developed are the difficulties associated with their purification. While commercial chromatography affinity resins, like staphylococcal superantigen-like 7 (SSL7) protein-containing resin, allow purification of IgAs from many animal species, these are not useful for murine IgAs. Because the mouse model is predominantly used for preclinical evaluation of IgA-based therapeutics, there is a need to develop an effective purification method for mouse IgA. Here, we adapted the sequence of a mouse IgA by mutating two amino acid residues in the fragment crystallizable (Fc) sequence to facilitate its purification on SSL7 resin. The mutated IgA Fc (hereafter referred to as IgA*) was then genetically fused to the variable domain of a llama heavy chain-only antibody (VHH) directed against the fusion protein of human respiratory syncytial virus (HRSV), resulting in VHH-IgA*, and transiently produced in infiltrated Nicotiana benthamiana leaves. These plant-produced mouse VHH-IgA* fusions were enriched by SSL7 affinity chromatography and were found to be functional in ELISA and could neutralize RSV in vitro, suggesting no detrimental effect of the mutation on their antigen-binding properties. This approach for the purification of murine IgA will facilitate downstream processing steps when designing innovative murine IgA-based fusions.

Tackling the recurrence of Clostridium difficile infection.

The pathogenesis of recurrent Clostridium difficile infection (CDI) is still poorly understood. The risk of recurrence is approximately 20% after an initial CDI episode and dramatically increases with subsequent CDI recurrences. Several factors may play a role in recurrent CDI (rCDI), including conditions influencing germination, metabolic pathways that influence toxin production of C. difficile, and the microbiota composition offering protection against colonization and disease caused by C. difficile. Paradoxically, the currently recommended treatment for acute symptomatic CDI, i.e. metronidazole or vancomycin, can cause modification of the intestinal flora. Indeed, administration of anti-CDI antibiotics leads to suppression of C. difficile, along with collateral damage of the protective intestinal microbiota and opening of a "window of vulnerability" for recurrence. Host factors also have a prominent role, including innate and acquired humoral immunity, i.e. passive antibodies administration or active vaccination as a prevention strategy. They play a crucial role in the protection against severe and recurrent CDI. The assessment of risk factors of recurrence and modeling prediction scores could help in preventing the troublesome experience of CDI recurrence. Six studies have methodologically assessed prediction scores for rCDI. However, the definition of recurrence was heterogeneous, external validation was often not performed, and immunological factors were often not considered. There is a need for further studies on the pathophysiology of recurrence to design models for prediction that are sound and applicable in clinical practice.

Streptococcus pyogenes translocates across an epithelial barrier.

Streptococcus pyogenes is a ß-hemolytic organism responsible for a wide variety of human diseases that commonly occur as self-limiting purulent diseases of the pharynx and skin. Although the occurrence of invasive infections by S. pyogenes is rare, mortality rates remain high even with progressive medical therapy. As a prerequisite for causing the severe invasive disease, S. pyogenes must invade underlying sterile tissues by translocating across the epithelial barrier. In this study, streptolysin S and SpeB were identified as the novel factors that facilitate bacterial translocation via degradation of intercellular junctions. Furthermore, we found that S. pyogenes exploits host plasminogen for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. Here, I would like to show our study on bacterial translocation across the epithelial barrier through paracellular route.

Enhancement of the cytotoxic activity of cytokine-induced killer cells transfected with IL3PE38KDEL gene against acute myeloid leukemia cells.

Cytokine-induced killer (CIK) cells, one of the feasible and effective methods of adoptive immunotherapy, have shown anti-leukemia activity in vivo and in vitro. But the strategy exhibits limited cytotoxic activity in clinical studies. In this study, CIK cells were transfected with an interleukin-3/Pseudomonas exotoxin gene (IL3PE38KDEL). RT-PCR and ELISA were used to verify the expression of IL3PE38KDEL in the transfected CIK cells. These cells released 1,186.7 ± 149.6 pg IL3PE38KDEL/10(4) cells over 48 h into the medium and the culture supernatant selectively killed IL3 receptor(IL3R)-positive HL60 cells, but not IL3R-negative K562 cells. Moreover, IL3PE38KDEL transfection did not influence phenotypes and cytokine production of CIK cells. Co-cultured with leukemia cells, IL3PE38KDEL transfected CIK cells showed enhanced cytotoxicity against IL3R-positive HL60 cells at all effector-to-target (E:T) ratios, but exerted a basal anti-leukemia activity against IL3R-negative K562 cells. Our findings demonstrate that IL3PE38KDEL gene transfection may be a novel strategy for improving anti-leukemia activity of CIK cells.

Sialic acid residues are essential for cell lysis mediated by leukotoxin from Aggregatibacter actinomycetemcomitans.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

Association between cytotoxic and invasive Pseudomonas aeruginosa and clinical outcomes in bacterial keratitis.

OBJECTIVES: To determine whether cytotoxic and invasive Pseudomonas aeruginosa strains differentially influence clinical presentation, outcomes, or therapeutic response in bacterial keratitis. METHODS: Pseudomonas aeruginosa isolates from the National Eye Institute-funded Steroids for Corneal Ulcers Trial were subtyped as cytotoxic or invasive strains. The main outcome measure compared between the 2 subtypes was change in visual acuity at 3 months using Huber robust regression, adjusting for topical corticosteroid treatment. RESULTS: Of 101 confirmed P aeruginosa isolates from the Steroids for Corneal Ulcers Trial, 74 had a classically cytotoxic or invasive genotype. While corneal ulcers caused by genotypically invasive P aeruginosa strains were associated at presentation with significantly better visual acuity than corneal ulcers caused by genotypically cytotoxic P aeruginosa strains when adjusting for the effect of ulcer location (P= .008), invasive ulcers had improved significantly less than cytotoxic ulcers at 3 months (0.35; 95% CI, 0.04-0.66 logMAR; P= .03 [3.5-line difference]). Compared with topical moxifloxacin alone, adjunctive treatment with topical corticosteroids was associated with significantly more improvement in visual acuity in the invasive subgroup (P= .04) but was associated with less improvement in visual acuity in the cytotoxic subgroup (P= .07). CONCLUSIONS: Rational profiling of differentially expressed virulence determinants (eg, cytotoxicity and invasiveness for P aeruginosa) could be used as a tool for decision making in the management of infections to optimize outcomes.

Structural and functional properties of staphylococcal superantigen-like protein 4.

Staphylococcus aureus is a prevalent and significant human pathogen. Among the repertoire of virulence factors produced by this bacterium are the 14 staphylococcal superantigen-like (SSL) proteins. SSL protein 4 (SSL4) is one member of this family and contains a highly conserved carbohydrate binding site also found in SSL2, SSL3, SSL5, SSL6, and SSL11. Recombinant SSL4(t), comprising amino acids 109 to 309 of Newman strain SSL4 (SSL4-Newman), has been shown to bind and be internalized by human granulocytes and macrophages in a sialic-acid (Sia)-dependent manner. SSL4(t) can compete with itself for cell binding, indicating that binding is target specific. A 2.5-Å-resolution crystal structure of SSL4(t) complexed with sialyl Lewis X (sLe(x)) [sLe(x)-Neu5Acα2-3Galß1-4(Fucα1-3)GlcNAc] revealed a similar binding site to SSL5 and SSL11. These data, along with data on SSL4(t) binding to a glycan array and biosensor analysis of sLe(x) and sialyllactosamine (sLacNac) binding are compared with those for SSL11. Although these proteins show great similarity in their carbohydrate binding sites, with a root mean square (RMS) difference between main chain atom positions of only 0.34 Å, these proteins differ in detail in their affinity for sLe(x) and sLacNac, as well as their glycan preference. Together with cell binding data, this shows how S. aureus produces multiple related proteins that target myeloid cells through specific sialyllactosamine-containing glycoproteins.

Sublytic concentrations of Staphylococcus aureus Panton-Valentine leukocidin alter human PMN gene expression and enhance bactericidal capacity.

CA-MRSA infections are often caused by strains encoding PVL, which can cause lysis of PMNs and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations, PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce O(2)(-) in response to fMLF, but unlike priming by LPS, this response did not require TLR signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL--a finding consistent with priming. Importantly, PMNs primed with PVL had an enhanced ability to bind/ingest and kill Staphylococcus aureus. Priming of PMNs with other agonists, such as IL-8 or GM-CSF, altered the ability of PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up-regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL-mediated priming of PMNs enhances the host innate immune response.

Ovarian hyperstimulation syndrome complicated by severe community-acquired pneumonia due to methicillin-resistant Staphylococcus aureus positive for Panton-Valentine leukocidin.

We report a case of severe ovarian hyperstimulation syndrome (OHSS) complicated by community-acquired methicillin-resistant Staphylococcus aureus-Panton-Valentine leukocidin positive (CAMRSA-PVL[+]) necrotizing pneumonia, sepsis and multiple organ failure (MOF) in a previously immunocompetent female. The patient required prolonged ventilatory support and intensive care unit (ICU) hospitalization. Multiple cavities and severely affected lung function persist 1 year after discharge.

Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

Relative contribution of three main virulence factors in Pseudomonas aeruginosa pneumonia.

OBJECTIVE: The pathogenesis and the outcome of Pseudomonas aeruginosa ventilator-acquired pneumonia depend on the virulence factors displayed by the bacteria as well as the host response. Thus, quorum sensing, lipopolysaccharide, and type 3 secretion system have each individually been shown to be important virulence systems in laboratory reference strains. However, the relative contribution of these three factors to the in vivo pathogenicity of clinically relevant strains has never been studied. We analyzed the virulence of 56 nonclonal Pseudomonas aeruginosa strains isolated from critically ill patients with ventilator-acquired pneumonia. To avoid the variation of human immune response, we used a murine model of pneumonia. The aim was to determine which virulence factor was the most important. SETTING: Research laboratory of a university. SUBJECTS: Male adult BALB/c mice. INTERVENTIONS: In vitro, the phenotype of each strain was established as to the expression of quorum sensing-regulated factors (elastase and pyocyanin), type 3 secretion system exotoxin secretion (Exotoxin U, S and/or T, or "nonsecreting"), and lipopolysaccharide O-antigen serotype. Strain pathogenicity was evaluated in vivo in a mouse model of acute pneumonia through lung injury assessment by measuring alveolar-capillary barrier permeability to proteins, lung wet/dry weight ratio, and bacterial dissemination. Associations were then sought between virulence system phenotypes and levels of lung injury. MEASUREMENTS AND MAIN RESULTS: In univariate analysis, elastase production, O11 serotype, and type 3 secretion system exotoxin secretion were associated with increased lung injury and exotoxin U was linked to an increase risk of bacteremia. In multivariate analysis, we observed that type 3 secretion system exotoxin secretion and to a lesser degree elastase production were associated with increased lung injury. CONCLUSION: In a murine model of pneumonia, our data suggest that type 3 secretion system and elastase are the most important virulence factors in clinically relevant P. aeruginosa strains.

Panton-Valentine leukocidin in the pathogenesis of community-associated methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that causes serious infectious diseases and was endemic in hospitals by the late 1960s. Beginning with its first report in the late 1990s, the rapid emergence of community-associated MRSA (CA-MRSA) worldwide responsible for a wide spectrum of diseases ranging from minor skin infections to fatal necrotizing pneumonia has been found in previously healthy individuals without established risk factors for MRSA acquisition. Recently, various virulence determinants unique to CA-MRSA have been uncovered, which explain how the pathogen spreads easily and causes severe CA-MRSA infections among humans. However, the role of Panton-Valentine leukocidin (PVL) in the pathogenesis of CA-MRSA infection is currently a matter of much debate because of conflicting data from epidemiologic studies of CA-MRSA infections and various murine disease models. Identifying specialized pathogenic traits of CA-MRSA and the concerted regulation of these factors remains a challenge that will foster development of vaccines and therapies designed to control CA-MRSA infections. This review focuses on the current status of molecular epidemiology associated with CA-MRSA in Taiwan and progresses toward understanding the enhanced virulence properties of CA-MRSA, with an emphasis on the role of Panton-Valentine leukocidin.