Transcriptome analysis unveils survival strategies of Streptococcus parauberis against fish serum

Streptococcus parauberis is an important bacterial fish pathogen that causes streptococcosis in a variety of fish species including the olive flounder. Despite its importance in the aquaculture industry, little is known about the survival strategy of S. parauberis in the host. Therefore, the objective of this study was to produce genome-wide transcriptome data and identify key factors for the survival of S. parauberis SPOF3K in its host. To this end, S. parauberis SPOF3K was incubated in olive flounder serum and nutrient-enriched media as a control. Although S. parauberis SPOF3K proliferated in both culture conditions, the transcriptomic patterns of the two groups were very different. Interestingly, the expression levels of genes responsible for the replication of an S. parauberis plasmid in the presence of olive flounder serum were higher than those in the absence of olive flounder serum, indicating that this plasmid may play an important role in the survival and proliferation of S. parauberis in the host. Several ATP-binding cassette transporters known to transport organic substrates (e.g., biotin and osmoprotectants) that are vital for bacterial survival in the host were significantly up-regulated in S. parauberis cultured in serum. In addition, groEL, dnaK operon, and members of the clp protease family, which are known to play important roles in response to various stressors, were up-regulated in S. parauberis incubated in serum, thus limiting damage and facilitating cellular recovery. Moreover, important virulence factors including the hyaluronic acid capsule (has operon), sortase A (srtA), C5a peptidase (scp), and peptidoglycan O-acetyltransferase (oatA) were significantly upregulated in S. paraubers in serum. These results indicate that S. paraubers can resist and evade the humoral immune responses of fish. The transcriptomic data obtained in this study provide a better understanding of the mode of action of S. parauberis in fish.

T4 Pili Promote Colonization and Immune Evasion Phenotypes of Nonencapsulated M4 Streptococcus pyogenes

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses. IMPORTANCE Group A Streptococcus (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets.

The Emergence of Successful Streptococcus pyogenes Lineages through Convergent Pathways of Capsule Loss and Recombination Directing High Toxin Expression

ABSTRACT Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s, respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (streptolysin O) loci conferring high expression of these toxins underpinned the success of these lineages, extension to other emm genotype lineages is unreported. The emergent emm89 lineage was characterized by five regions of homologous recombination additional to nga-slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesized that other leading genotypes may have undergone similar recombination events. We analyzed a longitudinal data set of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin genotype. We identified multiple associations between recombination at this locus and inactivating mutations within hasAB, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule and high expression of nga and slo may underpin the success of many emergent S. pyogenes lineages of different genotypes, triggering new pandemics, and could change the way S. pyogenes causes disease. IMPORTANCE Streptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for the majority of human infection in high-income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time.

The emergence of successfulStreptococcus pyogeneslineages through convergent pathways of capsule loss and recombination directing high toxin expression

Gene transfer and homologous recombination inStreptococcus pyogeneshas the potential to trigger the emergence of pandemic lineages, as exemplified by lineages ofemm1 andemm89 that emerged in the 1980s and 2000s respectively. Although near-identical replacement gene transfer events in thenga(NADase) andslo(Streptolysin O) locus conferring high expression of these toxins underpinned the success of these lineages, extension to otheremm-genotype lineages is unreported. The emergentemm89 lineage was characterised by five regions of homologous recombination additional tonga/slo, including complete loss of the hyaluronic acid capsule synthesis locushasABC,a genetic trait replicated in two other leadingemmtypes and recapitulated by otheremmtypes by inactivating mutations. We hypothesised that other leading genotypes may have undergone a similar recombination events. We analysed a longitudinal dataset of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection ofS. pyogenesgenomes representing 54 different genotypes, and found frequent evidence of recombination events at thenga-slolocus predicted to confer higher toxin expression. We identified multiple associations between recombination at this locus and inactivating mutations withinhasA/B,suggesting convergent evolutionary pathways in successful genotypes. This included common genotypesemm28 andemm87. The combination of no or low capsule, and high expression ofngaandslo,may underpin the success for many emergentS. pyogeneslineages of different genotypes, triggering new pandemics and could change the wayS. pyogenescauses disease.ImportanceStreptococcus pyogenesis a genetically diverse pathogen, with over 200 different genotypes defined byemmtyping, but only a minority of these genotypes are responsible for majority of human infection in high income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. AsS. pyogenespathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time.

One More Disguise in the Stealth Behavior of Streptococcus pyogenes

ABSTRACT The ability to hide in the animal kingdom is essential for survival; the same is true for bacteria . Streptococcus pyogenes is considered one of the more successful stealth bacteria in its production of a hyaluronic acid capsule that is chemically identical to the hyaluronic acid lining human joints. It has also acquired the capacity to enter eukaryotic cells to avoid the onslaught of the host’s immune defenses, as well as drugs. From this intracellular vantage point, it may remain dormant from days to weeks, only to cause disease again at a later time, perhaps causing a relapse in a drug-treated patient. We now learn that it is able to enter macrophages as well, enabling the Streptococcus to use this “Trojan horse” approach to be transported to distant sites in the body.

Genomic Characterization of a Pattern D Streptococcus pyogenesemm53Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity

ABSTRACTThe genome of an invasive skin-tropic strain (AP53) of serotype M53 group AStreptococcus pyogenes(GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales,viz.,emmgene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ∼30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS−) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS+strain. The collagen-binding protein transcriptsclBdiffered in the number of 5′-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (ΦAP53.2) contains the known virulence factor superantigen exotoxin gene tandemspeK-slaA. Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence.IMPORTANCEInfectious strains ofS. pyogenes(GAS) are classified by their serotypes, relating to the surface M protein, theemm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53.

The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule

The Gram-negative bacteriumPasteurella multocidais the causative agent of a number of economically important animal diseases, including avian fowl cholera. NumerousP. multocidavirulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that aP. multocidahfqmutant produces significantly less hyaluronic acid capsule during all growth phases and displays reducedin vivofitness. Transcriptional and proteomic analyses of thehfqmutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of thehfqmutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in thehfqmutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression ofP. multocidagenes, including the regulation of keyP. multocidavirulence factors, capsule, LPS, and filamentous hemagglutinin.

Trading Capsule for Increased Cytotoxin Production: Contribution to Virulence of a Newly Emerged Clade of emm89 Streptococcus pyogenes

ABSTRACTStrains ofemm89Streptococcus pyogeneshave become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125emm89strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3ngapromoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenesNADase) and SLO (streptolysin O). Second, all clade 3 strains lack thehasABClocus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report thatemm89strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3emm89S. pyogenes.IMPORTANCES. pyogenes(group A streptococcus [GAS]) causes pharyngitis (“strep throat”), necrotizing fasciitis, and other human infections. Serious infections caused byemm89S. pyogenesstrains have recently increased in frequency in many countries. Based on whole-genome sequence analysis of 1,125 strains recovered from patients on two continents, we discovered that a newemm89clone, termed clade 3, has two distinct genetic features compared to its predecessors: (i) absence of the genes encoding antiphagocytic hyaluronic acid capsule virulence factor and (ii) increased production of the secreted cytolytic toxins SPN and SLO.emm89S. pyogenesstrains with the clade 3 phenotype (absence of capsule and high expression of SPN and SLO) are highly virulent in mice. These findings provide new understanding of how new virulent clones emerge and cause severe infections worldwide. This newfound knowledge ofS. pyogenesvirulence can be used to help understand future epidemics and conduct new translational research.

Emergence of a New Highly Successful Acapsular Group A Streptococcus Clade of Genotype emm89 in the United Kingdom

ABSTRACTGroup AStreptococcus(GAS) genotypeemm89 is increasingly recognized as a leading cause of disease worldwide, yet factors that underlie the success of thisemmtype are unknown. Surveillance identified a sustained nationwide increase inemm89 invasive GAS disease in the United Kingdom, prompting longitudinal investigation of this genotype. Whole-genome sequencing revealed a recent dramatic shift in theemm89 population with the emergence of a new clade that increased to dominance over previousemm89 variants. Temporal analysis indicated that the clade arose in the early 1990s but abruptly increased in prevalence in 2008, coinciding with an increased incidence ofemm89 infections. Although standard variable typing regions (emmsubtype,teetype,softype, and multilocus sequence typing [MLST]) remained unchanged, uniquely the emergent clade had undergone six distinct regions of homologous recombination across the genome compared to the rest of the sequencedemm89 population. Two of these regions affected known virulence factors, the hyaluronic acid capsule and the toxins NADase and streptolysin O. Unexpectedly, and in contrast to the rest of the sequencedemm89 population, the emergent clade-associated strains were genetically acapsular, rendering them unable to produce the hyaluronic acid capsule. The emergent clade-associated strains had also acquired an NADase/streptolysin O locus nearly identical to that found inemm12 and modernemm1 strains but different from the rest of the sequencedemm89 population. The emergent clade-associated strains had enhanced expression of NADase and streptolysin O. The genome remodeling in the new clade variant and the resultant altered phenotype appear to have conferred a selective advantage over otheremm89 variants and may explain the changes observed inemm89 GAS epidemiology.IMPORTANCESudden upsurges or epidemic waves are common features of group A streptococcal disease. Although the mechanisms behind such changes are largely unknown, they are often associated with an expansion of a single genotype within the population. Using whole-genome sequencing, we investigated a nationwide increase in invasive disease caused by the genotypeemm89 in the United Kingdom. We identified a new clade variant that had recently emerged in theemm89 population after having undergone several core genomic recombination-related changes, two of which affected known virulence factors. An unusual finding of the new variant was the loss of the hyaluronic acid capsule, previously thought to be essential for causing invasive disease. A further genomic adaptation in the NADase/streptolysin O locus resulted in enhanced production of these toxins. Recombination-related genome remodeling is clearly an important mechanism in group AStreptococcusthat can give rise to more successful and potentially more pathogenic variants.