Survival In Whole Human Blood Research Articles

The Cell Wall Deacetylases Spy1094 and Spy1370 Contribute to Streptococcus pyogenes Virulence

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a strictly human pathogen that causes a wide range of diseases, including skin and soft tissue infections, toxic shock syndrome and acute rheumatic fever. We have recently reported that Spy1094 and Spy1370 of S. pyogenes serotype M1 are N-acetylglucosamine (GlcNAc) deacetylases. We have generated spy1094 and spy1370 gene deletion mutants in S. pyogenes and gain-of-function mutants in Lactococcus lactis. Similar to other cell wall deacetylases, our results show that Spy1094 and Spy1370 confer lysozyme-resistance. Furthermore, deletion of the genes decreased S. pyogenes virulence in a human whole blood killing assay and a Galleria mellonella (Greater wax moth) larvae infection model. Expression of the two genes in L. lactis resulted in increased lysozyme resistance and survival in whole human blood, and reduced survival of infected G. mellonella larvae. Deletion of the spy1370, but not the spy1094 gene, decreased resistance to the cationic antimicrobial peptide cecropin B, whereas both enzymes increased biofilm formation, probably resulting from the increase in positive charges due to deacetylation of the cell wall. In conclusion, Spy1094 and Spy1370 are important S. pyogenes virulence factors and might represent attractive targets for the development of antibacterial agents.

Loss of rpoE Encoding the δ-Factor of RNA Polymerase Impacts Pathophysiology of the Streptococcus pyogenes M1T1 Strain 5448.

Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and “housekeeping” genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.

Effects of Expression of Streptococcus pneumoniae PspC on the Ability of Streptococcus mitis to Evade Complement-Mediated Immunity

Streptococcus pneumoniae and Streptococcus mitis are genetically closely related and both frequently colonise the naso-oropharynx, yet S. pneumoniae is a common cause of invasive infections whereas S. mitis is only weakly pathogenic. We hypothesise that sensitivity to innate immunity may underlie these differences in virulence phenotype. We compared the sensitivity of S. pneumoniae and S. mitis strains to complement-mediated immunity, demonstrating S. mitis strains were susceptible to complement-mediated opsonophagocytosis. S. pneumoniae resistance to complement is partially dependent on binding of the complement regulator Factor H by the surface protein PspC. However, S. mitis was unable to bind factor H. The S. pneumoniae TIGR4 strain pspC was expressed in the S. mitis SK142 strain to create a S. mitis pspC+ strain. Immunoblots demonstrated the S. mitis pspC+ strain expressed PspC, and flow cytometry confirmed this resulted in Factor H binding to S. mitis, reduced susceptibility to complement and improved survival in whole human blood compared to the wild-type S. mitis strain. However, in mouse models the S. mitis pspC+ strain remained unable to establish persistent infection. Unlike S. pneumoniae strains, culture in serum or blood did not support increased CFU of the S. mitis strains. These results suggest S. mitis is highly sensitive to opsonisation with complement partially due to an inability to bind Factor H, but even when complement sensitivity was reduced by expression of pspC, poor growth in physiological fluid limited the virulence of S. mitis in mice.

LytR plays a role in normal septum formation and contributes to full virulence in Streptococcus suis

Streptococcus suis (S. suis) is a major zoonotic pathogen and is also responsible for variety of diseases in swine. LytR-CpsA-Psr (LCP) family proteins affect the biofilm formation and virulence of some Gram-positive bacteria, but we know nothing about their roles in S. suis. In this study, we constructed the LytR mutant and its revertant strains by natural transformation and verified them by PCR and western blot. We explored the effects of LytR on the cell morphology of S. suis. Transmission electron microscopic analysis showed that the mutant strain displayed aberrant septum placement with no obvious differences in capsular thickness. Crystal violet staining and laser-scanning confocal microscopy both revealed that LytR contributes to the biofilm formation of S. suis. The LytR mutant strain had reduced survival in whole human blood and was more sensitive to killing by polymorphonuclear leukocytes (PMNs). Furthermore, in a mouse infection model, the LytR mutant strain also exhibited significantly attenuated virulence and was more easily cleared in the blood. These results indicate that the LytR protein is involved in septum placement, biofilm formation and required for full virulence of S. suis during infection.

The general stress response of Staphylococcus aureus promotes tolerance of antibiotics and survival in whole human blood.

Staphylococcus aureus is a frequent cause of invasive human infections such as bacteraemia and infective endocarditis. These infections frequently relapse or become chronic, suggesting that the pathogen has mechanisms to tolerate the twin threats of therapeutic antibiotics and host immunity. The general stress response of S. aureus is regulated by the alternative sigma factor B (σB) and provides protection from multiple stresses including oxidative, acidic and heat. σB also contributes to virulence, intracellular persistence and chronic infection. However, the protective effect of σB on bacterial survival during exposure to antibiotics or host immune defences is poorly characterized. We found that σB promotes the survival of S. aureus exposed to the antibiotics gentamicin, ciprofloxacin, vancomycin and daptomycin, but not oxacillin or clindamycin. We also found that σB promoted staphylococcal survival in whole human blood, most likely via its contribution to oxidative stress resistance. Therefore, we conclude that the general stress response of S. aureus may contribute to the development of chronic infection by conferring tolerance to both antibiotics and host immune defences.

Incremental Contributions of FbaA and Other Impetigo-Associated Surface Proteins to Fitness and Virulence of a Classical Group A Streptococcal Skin Strain.

Group A streptococci (GAS) are highly prevalent human pathogens whose primary ecological niche is the superficial epithelial layers of the throat and/or skin. Many GAS strains with a strong tendency to cause pharyngitis are distinct from strains that tend to cause impetigo; thus, genetic differences between them may confer host tissue-specific virulence. In this study, the FbaA surface protein gene was found to be present in most skin specialist strains but largely absent from a genetically related subset of pharyngitis isolates. In an ΔfbaA mutant constructed in the impetigo strain Alab49, loss of FbaA resulted in a slight but significant decrease in GAS fitness in a humanized mouse model of impetigo; the ΔfbaA mutant also exhibited decreased survival in whole human blood due to phagocytosis. In assays with highly sensitive outcome measures, Alab49ΔfbaA was compared to other isogenic mutants lacking virulence genes known to be disproportionately associated with classical skin strains. FbaA and PAM (i.e., the M53 protein) had additive effects in promoting GAS survival in whole blood. The pilus adhesin tip protein Cpa promoted Alab49 survival in whole blood and appears to fully account for the antiphagocytic effect attributable to pili. The finding that numerous skin strain-associated virulence factors make slight but significant contributions to virulence underscores the incremental contributions to fitness of individual surface protein genes and the multifactorial nature of GAS-host interactions.

Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally “unintended” interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Is it true that plant-derived polyphenols are always beneficial for the human? In vitro study on Leonurus cardiaca extract properties in the context of the pathogenesis of Staphylococcus aureus infections.

The aim of the study was to determine whether Leonurus cardiaca L. herb extract (LCE) used at subinhibitory concentration modifies the characteristics of Staphylococcus aureus, which is important in the pathogenesis of invasive infections originating from the bloodstream, in a way favourable for the human host. Polyphenol-rich LCE, a common ingredient in pharmaceutical products used for various cardiovascular and nervous system disorders, had shown interesting antibacterial and antibiofilm properties in our previous studies. Our current findings indicate that the following S. aureus characteristics decreased, depending on the LCE concentration: (i) formation of aggregates in plasma, (ii) adherence to a fibrin-coated surface, (iii) staphylocoagulase-dependent plasma clotting, (iv) bacterial survival in whole human blood in an ex vivo model, (v) expression of cell surface protein A and (vi) synthesis of α-toxin. However, staphylococcal tolerance to exogenous hydrogen peroxide was enhanced after pre-incubation with LCE, possibly due to the increased activity of bacterial antioxidant enzymes, a possibility confirmed by the higher production of superoxide dismutase and slightly higher production of catalase. The use of LCE at sub-MIC in in vitro and ex vivo models resulted in the weakening of some important staphylococcal immunoprotective attributes but the strengthening of such virulence factors as those responsible for oxidative stress tolerance. Some of these results and the fact that LCE has direct anticoagulant properties, reflected in a reduced thrombin-dependent fibrinogen polymerization rate, suggest a risk of adverse effects, which could be important in the context of S. aureus survival in the host.

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

Nonencapsulated group A Streptococcus associated with human invasive disease (790.1)

Group A Streptococcus (GAS) is a leading human pathogen, causing 650,000 severe invasive diseases annually with 25% mortality. M1 GAS is the most frequently isolated serotype from invasive disease worldwide; however, a recent occurrence of invasive infections in Australia was attributed to M4 GAS. Here, we use a combined molecular genetics and bioinformatics approach to characterize 17 clinical M4 isolates associated with this invasive disease episode. The M4 isolates were classified into 3 pulsed‐field gel electrophoresis groups, and 2 multilocus sequence types. All isolates lacked hyaluronic acid (HA) capsule, a major antiphagocytic virulence factor, and whole genome sequence analysis of 2 isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Most isolates contained mutations within the covRS two‐component regulator, a hallmark of hyperinvasive GAS. Transformation of a plasmid expressing hasABC into M4 GAS restored capsule production. However, encapsulation neither promoted survival in whole human blood, nor enhanced virulence of wild‐type M4 GAS in a mouse model of systemic infection. Bioinformatic analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition by ancestral GAS. Together, these data demonstrate that nonencapsulated GAS is capable of causing life‐threatening invasive human infections.Grant Funding Source: National Health and Medical Research Council of Australia; National Institutes of Health

Full functional activity of SSL7 requires binding of both complement C5 and IgA

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a range of diseases, from local skin infections through to life-threatening illnesses such as toxic shock syndrome. S. aureus produces an assortment of molecules designed to evade or subvert the host immune system. One example is the 23 kDa staphylococcal superantigen-like protein 7 (SSL7) that simultaneously binds immunoglobulin A (IgA) and complement C5 to inhibit complement-mediated hemolytic and bactericidal activity. The avirulent bacterium Lactococcus lactis was engineered to express SSL7 so that its role in bacterial survival could be assessed without interference from other virulence factors. Expression of SSL7 by L. lactis led to significantly enhanced bacterial survival in whole human blood and prevented the membrane attack complex (C5b-9) forming on the cell wall. To further understand the mechanism of action of SSL7, the activity of wild-type SSL7 protein was compared with a panel of mutant proteins lacking the capacity to bind IgA, C5, or both IgA and C5. SSL7 potently inhibited in vitro chemotaxis of inflammatory myeloid cells in response to a pathogenic stimulus and when injected into mice, SSL7 blocked the migration of neutrophils into the peritoneum in response to an inoculum of heat-killed S. aureus. Mutagenesis of the C5-binding site on SSL7 abolished all inhibitory activity, while mutation of the IgA-binding site had only partial effects, indicating that while IgA binding enhances activity it is not essential. SSL7 is an important staphylococcal virulence factor with potent anti-inflammatory properties, which are mediated by targeting complement C5 and IgA.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus

The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.

Serotype- and strain- dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis

BackgroundThe Streptococcus pyogenes (group A streptococci, GAS) two-component signal transduction system CovRS has been described to be important for pathogenesis of this exclusively human bacterial species. If this system acts uniquely in all serotypes is currently unclear. Presence of serotype- or strain-dependent regulatory circuits and polarity is an emerging scheme in Streptococcus pyogenes pathogenesis. Thus, the contribution of the sensor kinase (CovS) of the global regulatory two-component signal transduction system CovRS on pathogenesis of several M serotypes was investigated.ResultsCovS mutation uniformly repressed capsule expression and hampered keratinocyte adherence in all tested serotypes. However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.ConclusionsThese data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent. This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.