PM2.5 impairs macrophage functions to exacerbate pneumococcus-induced pulmonary pathogenesis.

BACKGROUND: Pneumococcus is one of the most common human airway pathogens that causes life-threatening infections. Ambient fine particulate matter (PM) with aerodynamic diameter ≤ 2.5 µm (PM2.5) is known to significantly contribute to respiratory diseases. PM2.5-induced airway inflammation may decrease innate immune defenses against bacterial infection. However, there is currently limited information available regarding the effect of PM2.5 exposure on molecular interactions between pneumococcus and macrophages. RESULTS: PM2.5 exposure hampered macrophage functions, including phagocytosis and proinflammatory cytokine production, in response to pneumococcal infection. In a PM2.5-exposed pneumococcus-infected mouse model, PM2.5 subverted the pulmonary immune response and caused leukocyte infiltration. Further, PM2.5 exposure suppressed the levels of CXCL10 and its receptor, CXCR3, by inhibiting the PI3K/Akt and MAPK pathways. CONCLUSIONS: The effect of PM2.5 exposure on macrophage activity enhances pneumococcal infectivity and aggravates pulmonary pathogenesis.

Phosphorylation at the D53 but Not the T65 Residue of CovR Determines the Repression of rgg and speB Transcription in emm1- and emm49-Type Group A Streptococci.

CovR/CovS is a two-component regulatory system in group A Streptococcus and primarily acts as a transcriptional repressor. The D53 residue of CovR (CovRD53) is phosphorylated by the sensor kinase CovS, and the phosphorylated CovRD53 protein binds to the intergenic region of rgg-speB to inhibit speB transcription. Nonetheless, the transcription of rgg and speB is suppressed in covS mutants. The T65 residue of CovR is phosphorylated in a CovS-independent manner, and phosphorylation at the D53 and T65 residues of CovR is mutually exclusive. Therefore, how phosphorylation at the D53 and T65 residues of CovR contributes to the regulation of rgg and speB expression was elucidated. The transcription of rgg and speB was suppressed in the strain that cannot phosphorylate the D53 residue of CovR (CovRD53A mutant) but restored to levels similar to those of the wild-type strain in the CovRT65A mutant. Nonetheless, inactivation of the T65 residue phosphorylation in the CovRD53A mutant cannot derepress the rgg and speB transcription, indicating that phosphorylation at the T65 residue of CovR is not required for repressing rgg and speB transcription. Furthermore, trans complementation of the CovRD53A protein in the strain that expresses the phosphorylated CovRD53 resulted in the repression of rgg and speB transcription. Unlike the direct binding of the phosphorylated CovRD53 protein and its inhibition of speB transcription demonstrated previously, the present study showed that inactivation of phosphorylation at the D53 residue of CovR contributes dominantly in suppressing rgg and speB transcription.IMPORTANCE CovR/CovS is a two-component regulatory system in group A Streptococcus (GAS). The D53 residue of CovR is phosphorylated by CovS, and the phosphorylated CovRD53 binds to the rgg-speB intergenic region and acts as the transcriptional repressor. Nonetheless, the transcription of rgg and Rgg-controlled speB is upregulated in the covR mutant but inhibited in the covS mutant. The present study showed that nonphosphorylated CovRD53 protein inhibits rgg and speB transcription in the presence of the phosphorylated CovRD53in vivo, indicating that nonphosphorylated CovRD53 has a dominant role in suppressing rgg transcription. These results reveal the roles of nonphosphorylated CovRD53 in regulating rgg transcription, which could contribute significantly to invasive phenotypes of covS mutants.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Acidic stress enhances CovR/S-dependent gene repression through activation of the covR/S promoter in emm1-type group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) is a clinically important gram-positive bacterium that causes severe diseases with high mortality. Spontaneous mutations in genes encoding the CovR/CovS two-component regulatory system have been shown to derepress expression of virulence factors and are significantly associated with invasiveness of infections. Sensor kinase CovS senses environmental signals and then regulates the levels of phosphorylated CovR. In addition, CovS is responsible for survival of group A Streptococcus under acidic stress. How this system regulates the expression of CovR-controlled genes under acidic stress is not clear. This study shows that the expression of CovR-controlled genes, including hasA, ska, and slo, is repressed under acidic conditions by a CovS-dependent mechanism. Inactivation of CovS kinase activity or CovR protein phosphorylation derepresses the transcription of these genes under acidic conditions, suggesting that the phosphorylation of CovR is required for the repression of the CovR-controlled genes. Furthermore, the promoter activity of the covR/covS operon (pcov) was upregulated after 15min of incubation under acidic conditions. Replacement of pcov with a constitutively activated promoter abrogated the acidic-stress-dependent repression of the genes, indicating that the pH-dependent pcov activity is directly involved in the repression of CovR-controlled genes. In summary, the present study shows that inactivation of CovS not only derepresses CovR-controlled genes but also abrogates the acidic-stress-dependent repression of the genes; these phenomena may significantly increase bacterial virulence during infection.

Epidemiology Analysis of Streptococcus pyogenes in a Hospital in Southern Taiwan by Use of the Updated emm Cluster Typing System.

emm typing is the most widely used molecular typing method for the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). emm typing is based on a small variable region of the emm gene; however, the emm cluster typing system defines GAS types according to the nearly complete sequence of the emm gene. Therefore, emm cluster typing is considered to provide more information regarding the functional and structural properties of M proteins in different emm types of GAS. In the present study, 677 isolates collected between 1994 and 2008 in a hospital in southern Taiwan were analyzed by the emm cluster typing system. emm clusters A-C4, E1, E6, and A-C3 were the most prevalent emm cluster types and accounted for 67.4% of total isolates. emm clusters A-C4 and E1 were associated with noninvasive diseases, whereas E6 was significantly associated with both invasive and noninvasive manifestations. In addition, emm clusters D4, E2, and E3 were significantly associated with invasive manifestations. Furthermore, we found that the functional properties of M protein, including low fibrinogen-binding and high IgG-binding activities, were correlated significantly with invasive manifestations. In summary, the present study provides updated epidemiological information on GAS emm cluster types in southern Taiwan.

Capsule-deficient group A Streptococcus evades autophagy-mediated killing in macrophages.

The hyaluronic acid capsule is crucial in protecting group A Streptococcus (GAS) against phagocytic killing. However, there have been reported outbreaks caused by capsule-deficient GAS strains, and the mechanisms underlying their evasion of immune clearance remain unclear. This study demonstrated that the capsule-deficient mutant [Cap(-)] of the emm1 strain increased survival within phagocytic cells compared to the wild-type strain [Cap(+)]. Although both Cap(+) and Cap(-) strains exhibited similar abilities to disrupt the phagosome, only the Cap(+) strain was colocalized with lysosomes and acidified compartments in phagocytic cells, indicating its susceptibility to autophagosome elimination. In contrast, the Cap(-) mutant evaded the recognition of galectin-8 and ubiquitin, impairing selective autophagy-mediated elimination. These findings suggest that a deficiency in the capsule could impair the intracellular elimination of GAS in macrophages, revealing previously unknown aspects of the host's recognition of the GAS capsule in macrophages. IMPORTANCE: Group A Streptococcus (GAS) is a Gram-positive bacterium that causes diseases ranging from mild pharyngitis to severe necrotizing fasciitis. Phagocytic cells serve as the primary defense against bacterial infections, exhibiting remarkable efficiency in eliminating intracellular pathogens. The hyaluronic acid capsule is a critical virulence factor that contributes to the resistance of phagocytosis in GAS. Nevertheless, the outbreaks caused by GAS strains that lack the hyaluronic acid capsule have been reported, and the selective advantage of capsule-deficient strains during infection is not fully understood. This study showed that the autophagic adaptor proteins recognize the capsulated GAS strain but not the capsule-deficient mutant, indicating that the hyaluronic acid capsule could be the autophagic target in macrophages. These findings imply that the hyaluronic acid capsule of GAS actually enhances its elimination within phagocytic cells, subverting the understanding of the capsule in GAS pathogenesis.

Detection of toxigenic M1UK lineage group A Streptococcus clones in Taiwan.

BACKGROUND: A new sublineage of emm1 group A Streptococcus (GAS), M1UK, has emerged in Europe, North America, and Australia. Notably, a significant portion of emm1 isolates in Asia, particularly in Hong Kong and mainland China, acquired scarlet fever-associated prophages following the 2011 Hong Kong scarlet fever outbreak. However, the presence of the M1UK sublineage has not yet been detected in Asia. METHODS: This study included 181 GAS isolates (2011-2021). The emm type of these isolates were determined, and 21 emm1 isolates from blood or pleural fluid (2011-2021) and 10 emm1 isolates from throat swabs (2016-2018) underwent analysis. The presence of the scarlet fever-associated prophages and the specific single nucleotide polymorphisms of the M1UK clone were determined by polymerase chain reaction and the genome sequencing. RESULTS: The M1UK lineage strains from throat swab and blood samples were identified. One of the M1UK strain in Taiwan carried the scarlet fever-associated prophage and therefore acquired the ssa, speC, and spd1 toxin repertoire. Nonetheless, the increase of M1UK was not observed until 2021, and there was a reduction in the diversity of emm types in 2020-2021, possibly due to the COVID-19 pandemic restriction policies in Taiwan. CONCLUSIONS: Our results suggested that the M1UK lineage clone has introduced in Taiwan. In Taiwan, the COVID-19 restrictions were officially released in March 2023; therefore, it would be crucial to continuously monitor the M1UK expansion and its related diseases in the post COVID-19 era.

Conserved molecular chaperone PrsA stimulates protective immunity against group A Streptococcus.

Group A Streptococcus (GAS) is a significant human pathogen that poses a global health concern. However, the development of a GAS vaccine has been challenging due to the multitude of diverse M-types and the risk of triggering cross-reactive immune responses. Our previous research has identified a critical role of PrsA1 and PrsA2, surface post-translational molecular chaperone proteins, in maintaining GAS proteome homeostasis and virulence traits. In this study, we aimed to further explore the potential of PrsA1 and PrsA2 as vaccine candidates for preventing GAS infection. We found that PrsA1 and PrsA2 are highly conserved among GAS isolates, demonstrating minimal amino acid variation. Antibodies specifically targeting PrsA1/A2 showed no cross-reactivity with human heart proteins and effectively enhanced neutrophil opsonophagocytic killing of various GAS serotypes. Additionally, passive transfer of PrsA1/A2-specific antibodies conferred protective immunity in infected mice. Compared to alum, immunization with CFA-adjuvanted PrsA1/A2 induced higher levels of Th1-associated IgG isotypes and complement activation and provided approximately 70% protection against invasive GAS challenge. These findings highlight the potential of PrsA1 and PrsA2 as universal vaccine candidates for the development of an effective GAS vaccine.

RopB-regulated SpeB cysteine protease degrades extracellular vesicles-associated streptolysin O and bacterial proteins from group A Streptococcus.

Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the ropB mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.

Clostridium innocuum, an emerging pathogen that induces lipid raft-mediated cytotoxicity.

Clostridium innocuum is an emerging spore-forming anaerobe that is often observed in Clostridioides difficile-associated inflammatory bowel disease (IBD) exacerbations. Unlike C. difficile, C. innocuum neither produces toxins nor possesses toxin-encoding genetic loci, but is commonly found in both intestinal and extra-intestinal infections. Membrane lipid rafts are composed of dynamic assemblies of cholesterol and sphingolipids, allowing bacteria to gain access to cells. However, the direct interaction between C. innocuum and lipid rafts that confers bacteria the ability to disrupt the intestinal barrier and induce pathogenesis remains unclear. In this study, we investigated the associations among nucleotide-binding oligomerization domain containing 2 (NOD2), lipid rafts, and cytotoxicity in C. innocuum-infected gut epithelial cells. Our results revealed that lipid rafts were involved in C. innocuum-induced NOD2 expression and nuclear factor (NF)-κB activation, triggering an inflammatory response. Reducing cholesterol by simvastatin significantly dampened C. innocuum-induced cell death, indicating that the C. innocuum-induced pathogenicity of cells was lipid raft-dependent. These results demonstrate that NOD2 mobilization into membrane rafts in response to C. innocuum-induced cytotoxicity results in aggravated pathogenicity.

RopB represses the transcription of speB in the absence of SIP in group A Streptococcus.

RopB is a quorum-sensing regulator that binds to the SpeB-inducing peptide (SIP) under acidic conditions. SIP is known to be degraded by the endopeptidase PepO, whose transcription is repressed by the CovR/CovS two-component regulatory system. Both SIP-bound RopB (RopB-SIP) and SIP-free RopB (apo-RopB) can bind to the speB promoter; however, only RopB-SIP activates speB transcription. In this study, we found that the SpeB expression was higher in the ropB mutant than in the SIP-inactivated (SIP*) mutant. Furthermore, the deletion of ropB in the SIP* mutant derepressed speB expression, suggesting that apo-RopB is a transcriptional repressor of speB Up-regulation of PepO in the covS mutant degraded SIP, resulting in the down-regulation of speB We demonstrate that deleting ropB in the covS mutant derepressed the speB expression, suggesting that the speB repression in this mutant was mediated not only by PepO-dependent SIP degradation but also by apo-RopB. These findings reveal a crosstalk between the CovR/CovS and RopB-SIP systems and redefine the role of RopB in regulating speB expression in group A Streptococcus.

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection.

The Bacterial Markers of Identification of Invasive CovR/CovS-Inactivated Group A Streptococcus.

Necrotizing fasciitis is a severe infectious disease that results in significant mortality. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common bacterial pathogens of monomicrobial necrotizing fasciitis. The early diagnosis of necrotizing fasciitis is crucial; however, the typical cutaneous manifestations are not always presented in patients with GAS necrotizing fasciitis, which would lead to miss- or delayed diagnosis. GAS with spontaneous inactivating mutations in the CovR/CovS two-component regulatory system is significantly associated with destructive diseases such as necrotizing fasciitis and toxic shock syndrome; however, no specific marker has been used to identify these invasive clinical isolates. This study evaluated the sensitivity and specificity of using CovR/CovS-controlled phenotypes to identify CovR/CovS-inactivated isolates. Results showed that the increase of hyaluronic acid capsule production and streptolysin O expression were not consistently presented in CovS-inactivated clinical isolates. The repression of SpeB is the phenotype with 100% sensitivity of identifying in CovS-inactivated isolates among 61 clinical isolates. Nonetheless, this phenotype failed to distinguish RopB-inactivated isolates from CovS-inactivated isolates and cannot be utilized to identify CovR-inactivated mutant and RocA (Regulator of Cov)-inactivated isolates. In this study, we identified and verified that PepO, the endopeptidase which regulates SpeB expression through degrading SpeB-inducing quorum-sensing peptide, was a bacterial marker to identify isolates with defects in the CovR/CovS pathway. These results also inform the potential strategy of developing rapid detection methods to identify invasive GAS variants during infection. IMPORTANCE Necrotizing fasciitis is rapidly progressive and life-threatening; if the initial diagnosis is delayed, deep soft tissue infection can progress to massive tissue destruction and toxic shock syndrome. Group A Streptococcus (GAS) with inactivated mutations in the CovR/CovS two-component regulatory system are related to necrotizing fasciitis and toxic shock syndrome; however, no bacterial marker is available to identify these invasive clinical isolates. Inactivation of CovR/CovS resulted in the increased expression of endopeptidase PepO. Our study showed that the upregulation of PepO mediates a decrease in SpeB-inducing peptide (SIP) in the covR mutant, indicating that CovR/CovS modulates SIP-dependent quorum-sensing activity through PepO. Importantly, the sensitivity and specificity of utilizing PepO to identify clinical isolates with defects in the CovR/CovS pathway, including its upstream RocA regulator, were 100%. Our results suggest that identification of invasive GAS by PepO may be a strategy for preventing severe manifestation or poor prognosis after GAS infection.

Characterization of the phenotypes of methicillin- and vancomycin-susceptible Staphylococcus argenteus after vancomycin passages.

OBJECTIVES: Staphylococcus argenteus is generally more susceptible to antibiotic treatments than Staphylococcus aureus; however, the study showed that the daptomycin/vancomycin-resistant S. argenteus was isolated from a patient with repeated antibiotic treatments. In this study, the methicillin- and vancomycin-susceptible S. argenteus isolates were used to characterize the phenotypes of S. argenteus after vancomycin passages in vitro. METHODS: Eleven S. argenteus isolates were used for passaging under different concentrations of vancomycin. The minimal inhibitory concentration (MIC) of vancomycin was determined by the agar dilution assay, and the biofilm mass of the passaged variants was quantified by the crystal violet staining assay and observed under the confocal microscope. RESULTS: The MIC of vancomycin for eight of 11 S. argenteus isolates was increased from ≤2 µg/mL to ≤4-8 µg/mL after vancomycin passages. Two variants with the high-level vancomycin-intermediate (vancomycin MIC ≤8 µg/mL) phenotype were identified, and the parental strains of these variants did not have the heterogeneous vancomycin-intermediate population determined by the population profile analysis. Further, three S. argenteus isolates showed an increase in biofilm production and icaA transcription after the low-dose (2 µg/mL) vancomycin passages. CONCLUSIONS: S. argenteus is capable of acquiring a vancomycin-tolerant phenotype and/or converting to a strong biofilm producer after vancomycin passages, which could contribute to the decrease of their antibiotic susceptibility.

Clinical and microbiological characteristics of community-acquired Staphylococcus lugdunensis infections in Southern Taiwan.

Most Staphylococcus lugdunensis strains (49/59, 83%) were related to clinical infections, were susceptible to most antimicrobial agents with an overall oxacillin-resistant rate of 5% (3/58), and carried relatively great genetic diversity. Community-acquired infections (41/49, 84%) were dominant, often developed in patients with comorbidity, and had rather benign clinical courses without mortality.

Incidence and Effects of Acquisition of the Phage-Encoded ssa Superantigen Gene in Invasive Group A Streptococcus.

The acquisition of the phage-encoded superantigen ssa by scarlet fever-associated group A Streptococcus (Streptococcus pyogenes, GAS) is found in North Asia. Nonetheless, the impact of acquiring ssa by GAS in invasive infections is unclear. This study initially analyzed the prevalence of ssa+ GAS among isolates from sterile tissues and blood. Among 220 isolates in northern Taiwan, the prevalence of ssa+ isolates increased from 1.5% in 2008-2010 to 40% in 2017-2019. Spontaneous mutations in covR/covS, which result in the functional loss of capacity to phosphorylate CovR, are frequently recovered from GAS invasive infection cases. Consistent with this, Phostag western blot results indicated that among the invasive infection isolates studied, 10% of the ssa+ isolates lacked detectable phosphorylated CovR. Transcription of ssa is upregulated in the covS mutant. Furthermore, in emm1 and emm12 covS mutants, ssa deletion significantly reduced their capacity to grow in human whole blood. Finally, this study showed that the ssa gene could be transferred from emm12-type isolates to the emm1-type wild-type strain and covS mutants through phage infection and lysogenic conversion. As the prevalence of ssa+ isolates increased significantly, the role of streptococcal superantigen in GAS pathogenesis, particularly in invasive covR/covS mutants, should be further analyzed.

Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes.

Bacteria encounter oxidative stress by exposure to reactive oxygen species (ROS) present in the aerobic environment and during immune responses. In Streptococcus pyogenes, Dpr has been identified as a stress protein conferring resistance to hydrogen peroxide and multiple other stresses. The expression of Dpr is under perR (peroxide stress response regulator) control. However, the exact molecular mechanism of PerR regulation of Dpr is not clear. In this study, a perR deletion mutant was constructed by double cross-over mutagenesis. The profile of Dpr expression, performed by Western blot assay, revealed growth-phase dependency under normal culture conditions. Dpr expression decreased under iron-restricted conditions, whereas iron, zinc, nickel, and hydrogen peroxide induced its expression. The perR mutant does not induce Dpr as well when exposed to environmental signals. PerR binds the promoter region of dpr. Increased iron and hydrogen peroxide concentrations decreased PerR binding to the promoter region of dpr, suggesting that regulation of Dpr by environmental signals is mediated by PerR directly.

RocA Regulates Phosphatase Activity of Virulence Sensor CovS of Group A Streptococcus in Growth Phase- and pH-Dependent Manners.

The control of the virulence response regulator and sensor (CovR-CovS) two-component regulatory system in group A Streptococcus (GAS) strains regulates more than 15% of gene expression and has critical roles in invasive GAS infection. The membrane-embedded CovS has kinase and phosphatase activities, and both are required for modulating the phosphorylation level of CovR. Regulator of Cov (RocA) is a positive regulator of covR and also been shown to be a pseudokinase that interacts with CovS to enhance the phosphorylation level of CovR; however, how RocA modulates the activity of CovS has not been determined conclusively. Although the phosphorylation level of CovR was decreased in the rocA mutant in the exponential phase, the present study shows that phosphorylated CovR in the rocA mutant increased to levels similar to those in the wild-type strain in the stationary phase of growth. In addition, acidic stress, which is generally present in the stationary phase, enhanced the phosphorylation level of CovR in the rocA mutant. The phosphorylation levels of CovR in the CovS phosphatase-inactivated mutant and its rocA mutant were similar under acidic stress and Mg2+ (the signal that inhibits CovS phosphatase activity) treatments, suggesting that the phosphatase activity, but not the kinase activity, of CovS is required for RocA to modulate CovR phosphorylation. The phosphorylation level of CovR is crucial for GAS strains to regulate virulence factor expression; therefore, the growth phase- and pH-dependent RocA activity would contribute significantly to GAS pathogenesis.IMPORTANCE The emergence of invasive group A streptococcal infections has been reported worldwide. Clinical isolates that have spontaneous mutations or a truncated allele of the rocA gene (e.g., emm3-type isolates) are considered to be more virulent than isolates with the intact rocA gene (e.g., emm1-type isolates). RocA is a positive regulator of covR and has been shown to enhance the phosphorylation level of intracellular CovR regulator through the functional CovS protein. CovS is the membrane-embedded sensor and modulates the phosphorylation level of CovR by its kinase and phosphatase activities. The present study shows that the enhancement of CovR phosphorylation is mediated via the repression of CovS's phosphatase activity by RocA. In addition, we found that RocA acts dominantly on modulating CovR phosphorylation under neutral pH conditions and in the exponential phase of growth. The phosphorylation level of CovR is crucial for group A Streptococcus species to regulate virulence factor expression and is highly related to bacterial invasiveness; therefore, growth phase- and pH-dependent RocA activity and the sequence polymorphisms of rocA gene would contribute significantly to bacterial phenotype variations and pathogenesis.

Nicotinamide Increases Intracellular NAD+ Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

emm1/sequence type 28 strains of group A streptococci that express covR at early stationary phase are associated with increased growth and earlier SpeB secretion.

Streptococcus pyogenes (group A streptococcus [GAS]) is a versatile human pathogen, and emm1/sequence type 28 (ST28) is the most frequently isolated type from GAS infections. The emm1/ST28 strain is associated with necrotizing fasciitis and streptococcal toxic shock syndrome. Growth-phase regulation is one of the important regulatory mechanisms in GAS, which controls gene expression at restricted phases of growth. CovRS, a two-component regulatory system, is considered the regulator of streptococcal pyrogenic exotoxin B (SpeB) and is thought to be activated in the exponential phase of growth. In the present study, Northern hybridization analysis showed that 52% of the analyzed GAS strains expressed covR at the exponential phase, but 48% of the strains expressed covR at the early stationary phase of growth. Strains transcribing covR at the early stationary phase showed better growth and earlier SpeB expression than the other group of strains. Multilocus sequence typing and pulsed-field gel electrophoresis analysis showed only emm1/ST28 strains (which comprise a clonal cluster) were expressing covR at the early stationary phase of growth, indicating that emm1/ST28 strains have special characteristics which may be related to their worldwide distribution.