Cross-talk between the Aeromonas hydrophila type III secretion system and lateral flagella system

Aeromonas hydrophila is responsible for aeromonad septicaemia in fish, and gastroenteritis and wound infections in humans. The type III secretion system (T3SS) is utilized by aeromonads to inject protein effectors directly into host cells. One of the major genetic regulators of the T3SS in several bacterial species is the AraC-like protein ExsA. Previous studies have suggested a link between T3SS regulation and lateral flagella expression. The aim of this study was to determine the genetic regulation of the T3SS and its potential interaction with the lateral flagella system in A. hydrophila. To investigate the genes encoding the T3SS regulatory components exsA, exsD, exsC, and exsE were mutated and the activities of the T3SS promoters were measured in wild type and mutant backgrounds demonstrating a regulatory network. The Exs proteins were shown to interact with each other by BACTH assay and Far-Western Blot. The findings suggested a regulatory cascade in which ExsE was bound to the chaperone protein ExsC. When ExsC was free it sequestered the anti-activator ExsD thus stopping the inhibition of the T3SS master regulator ExsA allowing T3SS expression. The T3SS regulatory components were also shown to affect the expression of the lateral flagella system. The activities of the lateral flagella promoters were shown to be repressed by the absence of ExsD and ExsE, suggesting that the T3SS master regulator ExsA was a negative regulator of the lateral flagella system

Host Antimicrobial Peptides: the promise of new treatment strategies against Tuberculosis

Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective antitubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug-resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defense system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of antimycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel antitubercular chemotherapeutics

Characterization of transcription within sdr region of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen responsible for various infections in humans and animals. It causes localized and systemic infections, such as abscesses, impetigo, cellulitis, sepsis, endocarditis, bone infections, and meningitis. S. aureus virulence factors responsible for the initial contact with host cells (MSCRAMMs—microbial surface components recognizing adhesive matrix molecules) include three Sdr proteins. The presence of particular sdr genes is correlated with putative tissue specificity. The transcriptional organization of the sdr region remains unclear. We tested expression of the sdrC, sdrD, or sdrE genes in various in vitro conditions, as well as after contact with human blood. In this work, we present data suggesting a separation of the sdr region into three transcriptional units, based on their differential reactions to the environment. Differential reaction of the sdrD transcript to environmental conditions and blood suggests dissimilar functions of the sdr genes. SdrE has been previously proposed to play role in bone infections, whilst our results can indicate that sdrD plays a role in the interactions between the pathogen and human immune system, serum or specifically reacts to nutrients/other factors present in human blood

RocA Binds CsrS To Modulate CsrRS-Mediated Gene Regulation in Group A Streptococcus

Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus. Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus. Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation

PerR Confers Phagocytic Killing Resistance and Allows Pharyngeal Colonization by Group A Streptococcus

The peroxide response transcriptional regulator, PerR, is thought to contribute to virulence of group A Streptococcus (GAS); however, the specific mechanism through which it enhances adaptation for survival in the human host remains unknown. Here, we identify a critical role of PerR-regulated gene expression in GAS phagocytosis resistance and in virulence during pharyngeal infection. Deletion of perR in M-type 3 strain 003Sm was associated with reduced resistance to phagocytic killing in human blood and by murine macrophages in vitro. The increased phagocytic killing of the perR mutant was abrogated in the presence of the general oxidative burst inhibitor diphenyleneiodonium chloride (DPI), a result that suggests PerR-dependent gene expression counteracts the phagocyte oxidative burst. Moreover, an isogenic perR mutant was severely attenuated in a baboon model of GAS pharyngitis. In competitive infection experiments, the perR mutant was cleared from two animals at 24 h and from four of five animals by day 14, in sharp contrast to wild-type bacteria that persisted in the same five animals for 28 to 42 d. GAS genomic microarrays were used to compare wild-type and perR mutant transcriptomes in order to characterize the PerR regulon of GAS. These studies identified 42 PerR-dependent loci, the majority of which had not been previously recognized. Surprisingly, a large proportion of these loci are involved in sugar utilization and transport, in addition to oxidative stress adaptive responses and virulence. This finding suggests a novel role for PerR in mediating sugar uptake and utilization that, together with phagocytic killing resistance, may contribute to GAS fitness in the infected host. We conclude that PerR controls expression of a diverse regulon that enhances GAS resistance to phagocytic killing and allows adaptation for survival in the pharynx

A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection

Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection

M Protein and Hyaluronic Acid Capsule Are Essential for In Vivo Selection of covRS Mutations Characteristic of Invasive Serotype M1T1 Group A Streptococcus

The initiation of hyperinvasive disease in group A Streptococcus (GAS) serotype M1T1 occurs by mutation within the covRS two-component regulon (named covRS for control of virulence regulatory sensor kinase), which promotes resistance to neutrophil-mediated killing through the upregulation of bacteriophage-encoded Sda1 DNase. To determine whether other virulence factors contribute to this phase-switching phenomenon, we studied a panel of 10 isogenic GAS serotype M1T1 virulence gene knockout mutants. While loss of several individual virulence factors did not prevent GAS covRS switching in vivo, we found that M1 protein and hyaluronic acid capsule are indispensable for the switching phenotype, a phenomenon previously attributed uniquely to the Sda1 DNase. We demonstrate that like M1 protein and Sda1, capsule expression enhances survival of GAS serotype M1T1 within neutrophil extracellular traps. Furthermore, capsule shares with M1 protein a role in GAS resistance to human cathelicidin antimicrobial peptide LL-37. We conclude that a quorum of GAS serotype M1T1 virulence genes with cooperative roles in resistance to neutrophil extracellular killing is essential for the switch to a hyperinvasive phenotype in vivo

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract