Catabolite Sensing Regulatory Pathways in Group A Streptococcus Control Virulence and Host-Pathogen Interactions

As a pathogen Streptococcus pyogenes (Group A streptococcus) is remarkable for its ability to cause a wide range of diseases and infect hundreds of millions of people yearly. Group A streptococcus faces a unique challenge in its pathogenesis cycle in that humans are its only known reservoir, and colonization and infection commonly occur in the same tissue. Thus to persist within a specific host niche Group A streptococcus has developed a sizable arsenal of effectors and control mechanisms allowing it to combat and adapt to the host immune response. In most cases alteration of regulatory pathways controlling transcription of virulence factor genes leads to significant changes in the ability of Group A streptococcus to cause disease. Several environmental signals and regulators effecting expression of virulence factors have been identified. One of the most pervasive signals effecting streptococcal virulence factor transcription is growth phase. In virtually all described incidences, virulence factor transcript abundance is influenced by growth phase, yet specific signals and mechanisms of sensing growth phase have remained elusive. These studies were undertaken to study the mechanisms of growth phase dependent transcriptional regulation of Group A streptococcal virulence factors, to elucidate global changes in transcriptional control due to altered growth phase regulators, and to examine the role of growth phase regulation in host- pathogen interactions during streptococcal infection. In pursuit of these goals, we initially analyzed a growth phase controlled physiologic trait, the production of H2O2, linked to virulence in related pathogens. We identified the gene responsible for H2O2 production, lactate oxidase (lctO), and through transcriptional and biochemical analyses identified the carbon catabolite control protein (CcpA) as a central transcriptional regulator of growth phase in response to glucose availability. Inactivation of CcpA revealed global changes in virulence factor transcription and overlap with another recently identified carbohydrate regulator, LacD.1. Studies on these regulators indicated that while they respond to a common signal their effects on co- regulated genes are restricted to discrete times during streptococcal infection. Disruption of CcpA in particular led to attenuation of virulence and mis-regulation of a large contingent of virulence genes in infected tissues. Misregulation of target genes in the CcpA mutant correlated with an increase in host TNFα transcription, and occurred at times when wild type streptococci were causing maximal disease. Ablation of TNFα in the host led to an increase in local virulence in the CcpA deficient strain, and a switch from local to invasive disease during infection by wild type streptococci. In total, our studies have shown that growth phase regulation is comprised of multiple overlapping and organized pathways. Furthermore, functional growth phase regulation is necessary for Group A streptococcus to cause fully virulent disease, perhaps due to the interaction of growth phase regulated factors with host immune signaling pathways. Finally we have shown that TNFα is necessary to control localized infection and prevent dissemination to deeper tissues during infections by a streptococcal strain that normally causes localized non- spreading lesions. Understanding the mechanisms by which Group A streptococcus senses the host environment and interacts with the immune system is especially critical in studies of streptococcal pathogenesis and development of anti-streptococcal therapies. A broad spectrum streptococcal protective vaccine is unavailable and without treatment relatively minor streptococcal infections can progress to life threatening invasive disease or lead to highly disabling nonsuppurative sequelae. Our studies suggest that targeting growth phase sensitive regulatory pathways is a good strategy in combating streptococcal disease. In addition, in light of the use of many immunosuppressive therapies as treatments for various ailments, our results indicate the need for further investigations into the role TNFα in controlling human streptococcal diseases; patients receiving antiTNFα treatments may be at an increased risk for serious infections by Group A streptococcus. Further analysis and identification of specific growth phase controlled factors contributing to attenuation of CcpA deficient strains, identification of factors involved in alteration of host-pathogen interactions resulting in disparate TNFα responses, and elucidation of the mechanisms by which TNFα production leads to control of localized infection will provide insights into Gram positive bacterial infection, and the molecular mechanisms determining the outcome of Group A streptococcal disease

CcpA and LacD.1 Affect Temporal Regulation of Streptococcus pyogenes Virulence Genes ▿ †

Production of H2O2 follows a growth phase-dependent pattern that mimics that of many virulence factors of Streptococcus pyogenes. To gain greater insight into mechanisms coupling virulence factor expression to growth phase, we investigated the molecular basis for H2O2 generation and its regulation. Deletion of the gene encoding lactate oxidase (lctO) or culture in the presence of glucose eliminated H2O2 production, implicating carbohydrate regulation of lctO as a key element of growth phase control. In examining known carbohydrate-responsive regulators, deletion of the gene encoding CcpA but not that encoding LacD.1 resulted in both derepression and an uncoupling of lctO transcription from its growth phase pattern. Expanding this analysis to additional virulence factors demonstrated both negative (cfa, encoding CAMP factor) and positive (speB, encoding a cysteine protease) regulation by CcpA and that CcpA mutants were highly cytotoxic for cultured macrophages. This latter property resulted from enhanced transcription of the streptolysin S biogenesis operon. Examination of CcpA-promoter interactions using a DNA pull-down assay mimicking physiological conditions showed direct binding to the promoters of lctO and speB but not those of sagA. CcpA but not LacD.1 mutants were attenuated in a murine model of soft-tissue infection, and analysis of gene expression in infected tissue indicated that CcpA mutants had altered expression of lctO, cfa, and speB but not the indirectly regulated sagA gene. Taken together, these data show that CcpA regulates virulence genes via at least three distinct mechanisms and that disruption of growth phase regulation alters transcriptional patterns in infected tissues

Distinct Time-Resolved Roles for Two Catabolite-Sensing Pathways during Streptococcus pyogenes Infection▿ †

Many Gram-positive pathogens link the expression of virulence genes to the presence of carbon source substrates using overlapping pathways for global control of carbon catabolite regulation. However, how these pathways are integrated to control the behavior of the transcriptome in time- and compartment-specific patterns is typically not well understood. In the present study, global transcriptome profiling was used to determine the extent to which glucose alters gene expression in Streptococcus pyogenes (group A streptococcus) and the contributions of the CcpA and LacD.1 catabolite control pathways to the regulation of this response in vitro. This analysis revealed that the expression of as many as 15% of the genes examined was regulated and that CcpA and LacD.1 together contribute to the regulation of 60% of this subset. However, numerous patterns were observed, including both CcpA- and LacD.1-specific and independent regulation, coregulation, and regulation of genes by these pathways independently of glucose. In addition, CcpA and LacD.1 had antagonistic effects on most coregulated genes. To resolve the roles of these regulators during infection, the expression of selected transcripts representative of different regulatory patterns was examined in a murine model of soft tissue infection. This revealed distinct patterns of misregulation with respect to time in CcpA− versus LacD.1− mutants. Taken together, these data support an important role for carbohydrate in the regulation of the transcriptome in tissue and suggest that the CcpA and LacD.1 pathways are organized to function at different times during the course of an infection

Medical Malpractice and Diagnostic Radiology: Challenges and Opportunities

Medicolegal challenges in radiology are broad and impact both radiologists and patients. Radiologists may be affected directly by malpractice litigation or indirectly due to defensive imaging ordering practices. Patients also could be harmed physically, emotionally, or financially by unnecessary tests or procedures. As technology advances, the incorporation of artificial intelligence into medicine will bring with it new medicolegal challenges and opportunities. This article reviews the current and emerging direct and indirect effects of medical malpractice on radiologists and summarizes evidence-based solutions

Experimental Evolution In Vivo To Identify Selective Pressures during Pneumococcal Colonization

ABSTRACT Experimental evolution is a powerful technique to understand how populations evolve from selective pressures imparted by the surrounding environment. With the advancement of whole-population genomic sequencing, it is possible to identify and track multiple contending genotypes associated with adaptations to specific selective pressures. This approach has been used repeatedly with model species in vitro, but only rarely in vivo. Herein we report results of replicate experimentally evolved populations of Streptococcus pneumoniae propagated by repeated murine nasal colonization with the aim of identifying gene products under strong selection as well as the population genetic dynamics of infection cycles. Frameshift mutations in one gene, dltB, responsible for incorporation of d-alanine into teichoic acids on the bacterial surface, evolved repeatedly and swept to high frequency. Targeted deletions of dltB produced a fitness advantage during initial nasal colonization coupled with a corresponding fitness disadvantage in the lungs during pulmonary infection. The underlying mechanism behind the fitness trade-off between these two niches was found to be enhanced adherence to respiratory cells balanced by increased sensitivity to host-derived antimicrobial peptides, a finding recapitulated in the murine model. Additional mutations that are predicted to affect trace metal transport, central metabolism, and regulation of biofilm production and competence were also selected. These data indicate that experimental evolution can be applied to murine models of pathogenesis to gain insight into organism-specific tissue tropisms. IMPORTANCE Evolution is a powerful force that can be experimentally harnessed to gain insight into how populations evolve in response to selective pressures. Herein we tested the applicability of experimental evolutionary approaches to gain insight into how the major human pathogen Streptococcus pneumoniae responds to repeated colonization events using a murine model. These studies revealed the population dynamics of repeated colonization events and demonstrated that in vivo experimental evolution resulted in highly reproducible trajectories that reflect the environmental niche encountered during nasal colonization. Mutations impacting the surface charge of the bacteria were repeatedly selected during colonization and provided a fitness benefit in this niche that was counterbalanced by a corresponding fitness defect during lung infection. These data indicate that experimental evolution can be applied to models of pathogenesis to gain insight into organism-specific tissue tropisms