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.

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.

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-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.

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.

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.

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.

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.

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.

Rapid analysis of bacterial composition in prosthetic joint infection by 16S rRNA metagenomic sequencing.

OBJECTIVES: Prosthetic joint infection (PJI) is the most common cause of arthroplasty failure. However, infection is often difficult to detect by conventional bacterial cultures, for which false-negative rates are 23% to 35%. In contrast, 16S rRNA metagenomics has been shown to quantitatively detect unculturable, unsuspected, and unviable pathogens. In this study, we investigated the use of 16S rRNA metagenomics for detection of bacterial pathogens in synovial fluid (SF) from patients with hip or knee PJI. METHODS: We analyzed the bacterial composition of 22 SF samples collected from 11 patients with PJIs (first- and second-stage surgery). The V3 and V4 region of bacteria was assessed by comparing the taxonomic distribution of the 16S rDNA amplicons with microbiome sequencing analysis. We also compared the results of bacterial detection from different methods including 16S metagenomics, traditional cultures, and targeted Sanger sequencing. RESULTS: Polymicrobial infections were not only detected, but also characterized at different timepoints corresponding to first- and second-stage exchange arthroplasty. Similar taxonomic distributions were obtained by matching sequence data against SILVA, Greengenes, and The National Center for Biotechnology Information (NCBI). All bacteria isolated from the traditional culture could be further identified by 16S metagenomics and targeted Sanger sequencing. CONCLUSION: The data highlight 16S rRNA metagenomics as a suitable and promising method to detect and identify infecting bacteria, most of which may be uncultivable. Importantly, the method dramatically reduces turnaround time to two days rather than approximately one week for conventional cultures.Cite this article: M-F. Chen, C-H. Chang, C. Chiang-Ni, P-H. Hsieh, H-N. Shih, S. W. N. Ueng, Y. Chang. Rapid analysis of bacterial composition in prosthetic joint infection by 16S rRNA metagenomic sequencing. Bone Joint Res 2019;8:367-377. DOI: 10.1302/2046-3758.88.BJR-2019-0003.R2.

Current status of MALDI-TOF mass spectrometry in clinical microbiology.

Mass spectrometry (MS) is a type of analysis used to determine what molecules make up a sample, based on the mass spectrum that are created by the ions. Mass spectrometers are able to perform traditional target analyte identification and quantitation; however, they may also be used within a clinical setting for the rapid identification of bacteria. The causative agent in sepsis is changed over time, and clinical decisions affecting the management of infections are often based on the outcomes of bacterial identification. Therefore, it is essential that such identifications are performed quickly and interpreted correctly. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer is one of the most popular MS instruments used in biology, due to its rapid and precise identification of genus and species of an extensive range of Gram-negative and -positive bacteria. Microorganism identification by Mass spectrometry is based on identifying a characteristic spectrum of each species and then matched with a large database within the instrument. The present review gives a contemporary perspective on the challenges and opportunities for bacterial identification as well as a written report of how technological innovation has advanced MS. Future clinical applications will also be addressed, particularly the use of MALDI-TOF MS in the field of microbiology for the identification and the analysis of antibiotic resistance.

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.

Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells.

Group A streptococci (GAS) with spontaneous mutations in the CovR/CovS regulatory system are more invasive and related to severe manifestations. GAS can replicate inside phagocytic cells; therefore, phagocytic cells could serve as the niche to select invasive covS mutants. Nonetheless, the encapsulated covS mutant is resistant to phagocytosis. The fate of intracellular covS mutant in phagocytic cells and whether the intracellular covS mutant contributes to invasive infections are unclear. In this study, capsule-deficient (cap-) strains were utilized to study how intracellular bacteria interacted with phagocytic cells. Results from the competitive infection model showed that the cap- covS mutant had better survival fitness than the cap- wild-type strain in the PMA-activated U937 cells. In addition, the cap- covS mutant caused more cell damages than the cap- wild-type strain and encapsulated covS mutant. Furthermore, treatments with infected cells with clindamycin to inhibit the intracellular bacteria growth was more effective to reduce bacterial toxicity than utilized penicillin to kill the extracellular bacteria. These results not only suggest that the covS mutant could be selected from the intracellular niche of phagocytic cells but also indicating that inactivating or killing intracellular GAS may be critical to prevent invasive infection.

NAD-Glycohydrolase Depletes Intracellular NAD+ and Inhibits Acidification of Autophagosomes to Enhance Multiplication of Group A Streptococcus in Endothelial Cells.

Group A Streptococcus (GAS) is a human pathogen causing a wide spectrum of diseases, from mild pharyngitis to life-threatening necrotizing fasciitis. GAS has been shown to evade host immune killing by invading host cells. However, how GAS resists intracellular killing by endothelial cells is still unclear. In this study, we found that strains NZ131 and A20 have higher activities of NADase and intracellular multiplication than strain SF370 in human endothelial cells (HMEC-1). Moreover, nga mutants of NZ131 (SW957 and SW976) were generated to demonstrate that NADase activity is required for the intracellular growth of GAS in endothelial cells. We also found that intracellular levels of NAD+ and the NAD+/NADH ratio of NZ131-infected HMEC-1 cells were both lower than in cells infected by the nga mutant. Although both NZ131 and its nga mutant were trapped by LC3-positive vacuoles, only nga mutant vacuoles were highly co-localized with acidified lysosomes. On the other hand, intracellular multiplication of the nga mutant was increased by bafilomycin A1 treatment. These results indicate that NADase causes intracellular NAD+ imbalance and impairs acidification of autophagosomes to escape autophagocytic killing and enhance multiplication of GAS in endothelial cells.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

Highly prevalent emmSTG840.0 and emmSTC839.0 types of erythromycin non-susceptible group G Streptococcus isolated from bacteremia in southern Taiwan.

BACKGROUND/PURPOSE: Group G Streptococcus (GGS) infections in human have increased. Treatment relied on antibiotic therapy, including erythromycin. However, information regarding the dominant strains and erythromycin susceptibility in GGS bacteremia is limited. METHODS: A total of 134 GGS were isolated from patients with bacteremia in a university hospital of southern Taiwan during 1993-2010. The erythromycin susceptibility was determined by disc diffusion and agar dilution assays. The bacterial species was determined by MALDI-TOF. The presence of erythromycin-resistant genes and emm types were determined by polymerase chain reaction and sequence. The clonal spreading was analyzed by pulsed-field gel electrophoresis with SmaI or SgrAI digestion. RESULTS: The annual erythromycin non-susceptible rate varied, with an average of 40.3%. All erythromycin non-susceptible strains belonged to the Streptococcus dysgalactiae. No erythromycin non-susceptible strains belong to the anginosus group. The most prevalent erythromycin-resistant gene was mefA (57.4%), followed by ermB (37%), and ermA (3.7%). The N terminal hyper variable region of emm was sequenced to determine the emm type, and only S. dysgalactiae had the emm gene. The most prevalent emm types were emmSTG840.0 (17.2%), emmSTG485.0 (10.4%), and emmSTC839.0 (9.0%). 73% and 47% of the strains with only mefA and ermB belonged to emmSTG840.0 and emmSTC839.0 types, respectively. Pulsed-field gel electrophoresis showed that different clones of emmSTG840.0 and emmSTC839.0 strains were spread in this region during the 18 years of surveillance. CONCLUSION: Our data indicate that there were dominant emm types with erythromycin non-susceptibility in S. dysgalactiae isolated from bacteremia in Taiwan, and thus constant surveillance is warranted.