Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

Solvent-free electrically conductive Ag/ethylene vinyl acetate (EVA) composites for paper-based printable electronics.

Solvent-free electrically conductive composites have been applied to flexible electronics to obtain high electrical conductivity. However, some of the proposed composites have low electrical conductivities and are unable to meet the requirements of commercial printable electronics. In this study, solvent-free electrically conductive Ag/EVA (ethylene vinyl acetate) composites for paper-based printable electronics were prepared by a thermal melting method. The properties of these electrically conductive Ag/EVA composites, including particle sizes, morphologies and phase purities of the flake silver flake powders, were investigated using a particle size analyzer, scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The results showed that nanometer-thick flake silver flake powders with smooth and flat surfaces were made by the nanofilm transition technique. These obtained powders were able to form smooth face-to-face contacts, which facilitated the formation of an excellent conductive network in the conductive system. Dynamic mechanical analysis (DMA) was conducted to investigate the mechanical properties of EVA and Ag/EVA composites. A Fourier transformation infra-red (FTIR) spectrometer, laser micro-Raman spectrometer and thermogravimetric analyzer were used to analyze the organic functional groups, glass transition temperatures and thermal weight losses of the EVA resin and solvent-free electrically conductive composites. The solvent-free electrically conductive Ag/EVA composite, which contained 55 wt% of the as-prepared flake silver flake powders, was found to have an extremely low volume resistivity of 1.23 × 10-4 Ω cm as well as excellent bending performance and adhesion. These features indicate the great potential of these composites for application in printed electronics.

Hypervirulent Group A Streptococcus of Genotype emm3 Invades the Vascular System in Pulmonary Infection of Mice.

Natural mutations of the two-component regulatory system CovRS are frequently associated with invasive group A Streptococcus (GAS) isolates and lead to the enhancement of virulence gene expression, innate immune evasion, systemic dissemination, and virulence. How CovRS mutations enhance systemic dissemination is not well understood. A hypervirulent GAS isolate of the emm3 genotype, MGAS315, was characterized using a mouse model of pulmonary infection to understand systemic dissemination. This strain has a G1370T mutation in the sensor kinase covS gene of CovRS. Intratracheal inoculation of MGAS315 led to the lung infection that displayed extensive Gram staining at the alveolar ducts, alveoli, and peribronchovascular and perivascular interstitium. The correction of the covS mutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, the covS mutation allowed MGAS315 to disrupt and degrade the smooth muscle and endothelial layers of the blood vessels, directly contributing to systemic dissemination. It is concluded that hypervirulent emm3 GAS covS mutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination.

Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice.

Hypervirulent group A streptococcus (GAS) can inhibit neutrophil recruitment and cause systemic infection in a mouse model of skin infection. The purpose of this study was to determine whether platelet-activating factor acetylhydrolase Sse and streptolysin S (SLS) have synergistic contributions to inhibition of neutrophil recruitment and systemic infection in subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. Deletion of sse and sagA in MGAS315 synergistically reduced the skin lesion size and GAS burden in the liver and spleen. However, the mutants were persistent at skin sites and had similar growth factors in nonimmune blood. Thus, the low numbers of Δsse ΔsagA mutants in the liver and spleen were likely due to their reduction in the systemic dissemination. Few intact and necrotic neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and necrotic cells were present at the edge of Δsse mutant infection sites on day 1 and at the edge of and inside Δsse mutant infection sites on day 2. ΔsagA mutant infection sites had massive numbers of and few intact neutrophils at the edge and center of the infection sites, respectively, on day 1 and were full of intact neutrophils or necrotic cells on day 2. Δsse ΔsagA mutant infection sites had massive numbers of intact neutrophils throughout the whole infection site. These sse and sagA deletion-caused changes in the histological pattern at skin infection sites could be complemented. Thus, the sse and sagA deletions synergistically enhance neutrophil recruitment. These findings indicate that both Sse and SLS are required but that neither is sufficient for inhibition of neutrophil recruitment and systemic infection by hypervirulent GAS.

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence.

Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.

Contemporary Pharyngeal and Invasive emm1 and Invasive emm12 Group A Streptococcus Isolates Exhibit Similar In Vivo Selection for CovRS Mutants in Mice.

Group A Streptococcus (GAS) causes diverse infections ranging from common pharyngitis to rare severe invasive infections. Invasive GAS isolates can have natural mutations in the virulence regulator CovRS, which result in enhanced expression of multiple virulence genes, suppressed the expression of the protease SpeB, and increased virulence. It is believed that CovRS mutations arise during human infections with GAS carrying wild-type CovRS and are not transmissible. CovRS mutants of invasive GAS of the emm1 genotype arise readily during experimental infection in mice. It is possible that invasive GAS arises from pharyngeal GAS through rare genetic mutations that confer the capacity of mutated GAS to acquire covRS mutations during infection. The objective of this study was to determine whether contemporary pharyngeal emm1 GAS isolates have a reduced propensity to acquire CovRS mutations in vivo compared with invasive emm1 GAS and whether emm3, emm12, and emm28 GAS acquire CovRS mutants in mouse infection. The propensity of invasive and pharyngeal emm1 and invasive emm3, emm12, and emm28 SpeBA+ isolates to acquire variants with the SpeBA- phenotype was determined during subcutaneous infection of mice. The majority of both invasive and pharyngeal emm1 SpeBA+ isolates and two of three emm12 isolates, but not emm3 and emm28 isolates, were found to acquire SpeBA- variants during skin infection in mice. All analyzed SpeBA- variants of emm1 and emm12 GAS from the mouse infection acquired covRS mutations and produced more platelet-activating factor acetylhydrolase SsE. Thus, contemporary invasive and pharyngeal emm1 GAS isolates and emm12 GAS have a similar capacity to acquire covRS mutations in vivo. The rarity of severe invasive infections caused by GAS does not appear to be attributable to a reduced ability of pharyngeal isolates to acquire CovRS mutations.

Controlled release behaviour and antibacterial effects of antibiotic-loaded titania nanotubes.

Bacterial infections have been identified as the main cause of orthopaedic implant failure. Owing to their high antibiotic delivery efficiency, titania nanotubes loaded with antibiotics constitute one of the most promising strategies for suppressing bacterial infections. However, it is difficult to control the drug-release behaviour of such nanotubes. Although sealing the nanotubes with a polymer solution provides sustained release effects to a certain extent, it inevitably influences their initial antibacterial activity. This study reports on the controlled release of gentamicin sulphate (GS) from titania nanotube surfaces whereby their initial antibacterial activity remains unaffected. Titania nanotubes were fabricated via electrochemical anodization and loaded with GS through physical adsorption. Experimental results showed that this loading method is feasible and efficient. The GS-loaded titania nanotubes were further covered by a thin film comprising a mixture of GS and chitosan (GSCH). The release kinetics confirmed that the drug release could be controlled by this thin film. Moreover, such a film was shown to not only inhibit initial bacterial adherence owing to its strong antibacterial properties but also enhance cell viability. Thus, GS-loaded titania nanotubes coated with GSCH have considerable potential as biomaterials for preventing initial release and peri-implant infection in the field of orthopaedics.

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms.

Invasive M1T1 group A Streptococcus (GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in Δsda1 mutants with (5448 Δsda1(M+) strain) and without (5448 Δsda1(M-) strain) M protein production. The 5448 Δsda1(M+) strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 Δsda1(M-) strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues.

The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covSwt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covSwt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.

A Neutralizing Monoclonal IgG1 Antibody of Platelet-Activating Factor Acetylhydrolase SsE Protects Mice against Lethal Subcutaneous Group A Streptococcus Infection.

Group A Streptococcus (GAS) can cause life-threatening invasive infections, including necrotizing fasciitis. There are no effective treatments for severe invasive GAS infections. The platelet-activating factor (PAF) acetylhydrolase SsE produced by GAS is required for invasive GAS to evade innate immune responses and to invade soft tissues. This study determined whether the enzymatic activity of SsE is critical for its function in GAS skin invasion and inhibition of neutrophil recruitment and whether SsE is a viable target for immunotherapy for severe invasive GAS infections. An isogenic derivative of M1T1 strain MGAS5005 producing SsE with an S178A substitution (SsE(S178A)), an enzymatically inactive SsE mutant protein, was generated. This strain induced higher levels of neutrophil infiltration and caused smaller lesions than MGAS5005 in subcutaneous infections of mice. This phenotype is similar to that of MGAS5005 sse deletion mutants, indicating that the enzymatic activity of SsE is critical for its function. An anti-SsE IgG1 monoclonal antibody (MAb), 2B11, neutralized the PAF acetylhydrolase activity of SsE. Passive immunization with 2B11 increased neutrophil infiltration, reduced skin invasion, and protected mice against MGAS5005 infection. However, 2B11 did not protect mice when it was administered after MGAS5005 inoculation. MGAS5005 induced vascular effusion at infection sites at early hours after GAS inoculation, suggesting that 2B11 did not always have access to infection sites. Thus, the enzymatic activity of SsE mediates its function, and SsE has the potential to be included in a vaccine but is not a therapeutic target. An effective MAb-based immunotherapy for severe invasive GAS infections may need to target virulence factors that are critical for systemic survival of GAS.

Neutrophils select hypervirulent CovRS mutants of M1T1 group A Streptococcus during subcutaneous infection of mice.

Pathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group A Streptococcus (GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeB(A-)]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeB(A-) variants (SpeB(A-)%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeB(A-)% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2(-/-) mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2(-/-) mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeB(A-) variants in CXCR2(-/-) mice. One randomly chosen SpeB(A-) mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeB(A-) variants from a mouse all had nonsynonymous covRS mutations that resulted in the SpeB(A-) phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneous covRS mutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants.

The sagA/pel locus does not regulate the expression of the M protein of the M1T1 lineage of group A Streptococcus.

Altered expression of Group A Streptococcus (GAS) virulence factors, including the M protein, can result as a consequence of spontaneous genetic changes that occur during laboratory and animal passage. Occurrence of such secondary mutations during targeted gene deletion could confound the interpretation of effects attributable to the function of the gene being investigated. Contradicting reports on whether the sagA/pel locus regulates the M protein-encoding emm might be due to inconsistent occurrence of mutations unrelated with sagA. This study examined the possibility that altered emm expression observed in association with sagA/pel deletion mutants is artifactual. sagA deletion mutants (MGAS2221ΔsagA) of M1T1 isolate MGAS2221 obtained using liquid broth for GAS growth during the deletion process had diminished emm transcription and no detectable M protein production. In contrast, a ΔsagA mutant of another closely genetically related M1T1 isolate had normal emm expression. The sagB gene does not regulate emm; however, one of three MGAS2221ΔsagB mutants had diminished emm expression. The emm regulator mga was downregulated in these M protein expression-negative strains. These results argue that sagA deletion does not directly cause the downregulation of emm expression. Indeed, two MGAS2221ΔsagA mutants obtained using agar plates for GAS growth during the deletion process both had normal emm expression. We conclude that the sagA/pel locus does not regulate emm expression in the M1T1 lineage and provide a protocol for targeted gene deletion that we find less prone to the generation of mutants exhibiting downregulation in emm expression.

Regulation of inhibition of neutrophil infiltration by the two-component regulatory system CovRS in subcutaneous murine infection with group A streptococcus.

Hypervirulent invasive group A streptococcus (GAS) isolates inhibit neutrophil infiltration more than pharyngitis isolates do, and the molecular basis of this difference is not well understood. This study was designed to first determine whether natural null mutation of the two-component regulatory system CovRS is responsible for the enhancement of the inhibition of neutrophil recruitment seen in hypervirulent GAS. Next, we examined the role of CovRS-regulated interleukin-8/CXC chemokine peptidase (SpyCEP), C5a peptidase (ScpA), and platelet-activating factor acetylhydrolase (SsE) in the enhanced innate immune evasion. Invasive isolate MGAS5005 induces less neutrophil infiltration and produced a greater lesion area than pharyngitis isolate MGAS2221 in subcutaneous infections of mice. It is known that MGAS5005, but not MGAS2221, has a natural 1-bp deletion in the covS gene. Replacement of covS(Δ1bp) in MGAS5005 with wild-type covS resulted in the MGAS2221 phenotype. Deletion of covS from MGAS2221 resulted in the MGAS5005 phenotype. Tests of single, double, and triple deletion mutants of the MGAS5005 sse, spyCEP, and scpA genes found that SsE plays a more important role than SpyCEP and ScpA in the inhibition of neutrophil recruitment and that SsE, SpyCEP, and ScpA do not have synergistic effects on innate immune evasion by MGAS5005. Deletion of sse, but not spyCEP or scpA, of MGAS2221 enhances neutrophil recruitment. Thus, covS null mutations can cause substantial inhibition of neutrophil recruitment by enhancing the expression of the chemoattractant-degrading virulence factors, and SsE, but not SpyCEP or ScpA, is required for CovRS-regulated GAS inhibition of neutrophil infiltration.

Group A Streptococcus secreted esterase hydrolyzes platelet-activating factor to impede neutrophil recruitment and facilitate innate immune evasion.

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse(MGAS5005)) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K(M) and k(cat) of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse(MGAS5005) infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.