New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates

A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcrip-tome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications. IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.Peer reviewe

Population Genomic Molecular Epidemiological Study of Macrolide-Resistant Streptococcus pyogenes in Iceland, 1995 to 2016: Identification of a Large Clonal Population with a pbp2x Mutation Conferring Reduced In Vitro β-Lactam Susceptibility

Publisher's version (útgefin grein)Resistance to macrolide antibiotics is a global concern in the treatment of Streptococcus pyogenes (group A Streptococcus [GAS]) infections. In Iceland, since the detection of the first macrolide-resistant isolate in 1998, three epidemic waves of macrolide-resistant GAS infections have occurred, with peaks in 1999, 2004, and 2008. We conducted whole-genome sequencing of all 1,575 available GAS macrolide-resistant clinical isolates of all infection types collected at the national reference laboratory in Reykjavik, Iceland, from 1998 to 2016. Among 1,515 erythromycin-resistant isolates, 90.3% were of only three emm types, emm4 (n = 713), emm6 (n = 324), and emm12 (n = 332), with each being predominant in a distinct epidemic peak. The antibiotic efflux pump genes, mef(A) and msr(D), were present on chimeric mobile genetic elements in 99.3% of the macrolide-resistant isolates of these emm types. Of note, in addition to macrolide resistance, virtually all emm12 isolates had a single amino acid substitution in penicillin-binding protein PBP2X that conferred a 2-fold increased penicillin G and ampicillin MIC among the isolates tested. We conclude that each of the three large epidemic peaks of macrolide-resistant GAS infections occurring in Iceland since 1998 was caused by the emergence and clonal expansion of progenitor strains, with macrolide resistance being conferred predominantly by inducible Mef(A) and Msr(D) drug efflux pumps. The occurrence of emm12 strains with macrolide resistance and decreased beta-lactam susceptibility was unexpected and is of public health concern.This study was supported in part by the Fondren Foundation, Houston Methodist Hospital and Research Institute, and National Institutes of Health grants AI139369 and AI146771 (to J.M.M.).Peer Reviewe

Transcriptome Remodeling Contributes to Epidemic Disease Caused by the Human Pathogen Streptococcus pyogenes

For over a century, a fundamental objective in infection biology research has been to understand the molecular processes contributing to the origin and perpetuation of epidemics. Divergent hypotheses have emerged concerning the extent to which environmental events or pathogen evolution dominates in these processes. Remarkably few studies bear on this important issue. Based on population pathogenomic analysis of 1,200 Streptococcus pyogenes type emm89 infection isolates, we report that a series of horizontal gene transfer events produced a new pathogenic genotype with increased ability to cause infection, leading to an epidemic wave of disease on at least two continents. In the aggregate, these and other genetic changes substantially remodeled the transcriptomes of the evolved progeny, causing extensive differential expression of virulence genes and altered pathogen-host interaction, including enhanced immune evasion. Our findings delineate the precise molecular genetic changes that occurred and enhance our understanding of the evolutionary processes that contribute to the emergence and persistence of epidemically successful pathogen clones. The data have significant implications for understanding bacterial epidemics and for translational research efforts to blunt their detrimental effects. IMPORTANCE The confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease.Peer reviewe

Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae

The contribution of competence system to the virulence of Streptococcus pneumoniae

Competence induction in Streptococcus pneumoniae (pneumococcus) is initiated by the binding of the 17-amino acid competence stimulating peptide (CSP) to its membrane-associated histidine sensor kinase ComD. ComD autophosphorylates and subsequently phosphorylates and activates its cognate response regulator ComE. CSP-ComDE triggers the expression of 24 “early” competence genes and approximately 80 “late” competence genes. Some late competence genes encode effectors for genetic transformation. In recent years, the competence regulon of S. pneumoniae has been shown to cross regulate virulence. For example, Lau et al reported that the loss of function in ComB, an accessory protein to the ComA ABC transporter required for the export of CSP, as well as loss of function in the ComD, attenuate the ability of S. pneumoniae to cause pneumonia and bacteremia in mice. Because these studies suggest that competence regulon is not only essential for genetic transformation, but also for virulence, I sought a strategy to suppress pneumococcal competence development as a mean to reduce horizontal gene transfer as well as virulence. Of all the steps required for the development of competence for genetic transformation, the only one that occurs extracellularlly is the interaction between CSP and ComD. I hypothesized that blocking CSP-ComD interaction could effectively inhibit the induction of competence system. Previous studies have shown that the variable central region of CSP determines its receptor specificity. However, the roles of the conserved amino acid residues on both the N- and C-termini are unknown. I examined if amino acid substitution or deletion of these conserved residues could generate analogs that inhibit competence induction by wild-type CSP. I demonstrate that one of the CSP analogs, CSP1-E1A, competitively inhibits the ability of CSP1 to induce genetic transformation in a concentration dependent manner. I also demonstrate that CSP1-E1A successfully attenuates the expression of two virulence factors, CbpD and LytA during competence induction in a concentration-dependent manner. Inhibition of LytA and CbpD by CSP1-E1A reveals the possibility of using this peptide analog to attenuate pneumococcal infection. I examined whether CSP1-E1A could attenuate the virulence of S. pneumoniae D39 using a mouse model of acute pneumonia. CSP1-E1A significantly reduced the mortality rate as well as the kinetic of death infected mice. These results suggest that CSP1-E1A is able to attenuate virulence mechanisms of pneumococcus regulated by the competence regulon. One important feature of pneumococcal DNA uptake during competence is the degradation of one strand of donor DNA by the pneumococcal membrane nuclease EndA. EndA-mediated DNA degradation and small DNA fragment release were used as indicators of competence development. Interestingly, EndA has been identified as a virulence factor, most probably by degrading the neutrophil extracellular traps (NETs) and facilitate pneumococcal dissemination. I hypothesized that the nucleolytic activity of EndA during competence development is important for virulence. Contrary to my expectations, my experimental results demonstrate that EndA-mediated degradation of extracellular DNA is independent of the competence state of pneumococcus. S. pneumoniae mutants with deletion in genes essential for competence induction and regulation have extracellular nuclease activity comparable to their parental wild-type. In addition, nuclease activity of EndA is independent of LytA-mediated partial cell lysis, which may release the endonuclease into DNA-rich environments. Based on these results, I have proposed the existence of two distinct physiological states of EndA: the “competent state” and the “constitutive state”. During the competent state, a portion of EndA molecules is recruited by the competent apparatus to degrade one strand of the DNA, allowing the uptake of the remaining single stranded DNA for integration. During the “constitutive state”, EndA degrades extracellular DNA randomly. This portion of EndA activity is primarily responsible for the degradation of (NETs, virulence and bacterial dissemination. In addition, my data demonstrate that EndA molecules are secreted into the environment, and secreted EndA contributes substantially to “constitutive state” nucleolytic activity. Finally, the nuclease inhibitor aurintricarboxylic acid attenuates EndA-mediated genetic transformation as well as degradation of NETs. Since EndA is essential for both virulence and genetic transformation, it may serve as an interesting drug target to attenuate pneumococcal infection and inhibit horizontal gene transfer. A key event of competence development in S. pneumoniae is the expression of pneumococcal alternative sigma factor ComX. ComX initiates the expression of 80 “late” competence genes in pneumococcal cells competent for genetic transformation. I hypothesized that the expression of ComX-regulated genes is beneficial to the fitness of S. pneumoniae during host infection. In this study, I conducted a systematic deletion study on the contribution ComX-regulated late genes to mouse infection. I showed that 13 mutants with deletions of ComX-regulated genes are attenuated in at least one model of mouse infection. For some late competence genes, their specific expression during competence contributes to bacterial fitness. While for some other ComX regulated genes, their constitutive baseline expression is important for bacterial fitness. Finally, inactivating dprA, a gene essential for competence shut off, severely attenuates the virulence of pneumococcus, indicating competence needs to be properly regulated during host infection. Collectively, these experimental data suggest that the competence regulon of S. pneumoniae plays crucial rules in both genetic transformation as well as fitness and survival in the hosts

Saturated alanine scanning mutagenesis of the pneumococcus competence stimulating peptide identifies analogs that inhibit genetic transformation.

Antibiotic resistance is a major challenge to modern medicine. Intraspecies and interspecies dissemination of antibiotic resistance genes among bacteria can occur through horizontal gene transfer. Competence-mediated gene transfer has been reported to contribute to the spread of antibiotic resistance genes in Streptococcus pneumoniae. Induction of the competence regulon is mediated by a 17-amino acid peptide pheromone called the competence stimulating peptide (CSP). Thus, synthetic analogs that competitively inhibit CSPs may reduce horizontal gene transfer. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on CSP1 to screen for analogs that disable genetic transformation in S. pneumoniae. Substitution of the glutamate residue at the first position created analogs that could competitively inhibit CSP1-mediated competence development in a concentration-dependent manner. Additional substitutions of the negatively-charged glutamate residue with amino acids of different charge, acidity and hydrophobicity, as well as enantiomeric D-glutamate, generated analogs that efficiently outcompeted CSP1, suggesting the importance of negative charge and enantiomericity of the first glutamate residue for the function of CSP1. Collectively, these results indicate that glutamate residue at the first position is important for the ability of CSP1 to induce ComD, but is dispensable for the peptide to bind the receptor. Furthermore, these results demonstrate the potential applicability of competitive CSP analogs to control horizontal transfer of antibiotic resistance genes in S. pneumoniae

Competence-independent activity of pneumococcal EndA [corrected] mediates degradation of extracellular dna and nets and is important for virulence.

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection

Correction: Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence.

[This corrects the article on p. e70363 in vol. 8.]