Group Of Virulence Factors Research Articles

Molecular epidemiology and virulence factors of group B Streptococcus in South Korea according to the invasiveness

BackgroundGroup B Streptococcus (GBS) causes invasive infections in newborns and elderly individuals, but is a noninvasive commensal bacterium in most immunocompetent people. Recently, the incidence of invasive GBS infections has increased worldwide, and there is growing interest in the molecular genetic characteristics of invasive GBS strains. Vaccines against GBS are expected in the near future. Here, we aimed to analyze the molecular epidemiology of GBS according to the invasiveness in South Korea.MethodsWe analyzed GBS isolates collected and stored in two hospitals in South Korea between January 2015 and December 2020. The invasiveness of these isolates was determined via a retrospective review of clinical episodes. Totally, 120 GBS isolates from 55 children and 65 adults were analyzed. Serotype and sequence type (ST) were determined using multiplex polymerase chain reaction (PCR) and multilocus sequence typing, respectively. Fourteen virulence factor-encoding genes of GBS were analyzed using multiplex PCR.ResultsForty one (34.2%) were invasive infection-related GBS isolates (iGBS). The most frequently detected serotype was III (39/120, 32.5%), and it accounted for a high proportion of iGBS (21/41, 51.2%). The most frequent ST was ST19 (18/120, 15.0%), followed by ST2 (17/120, 14.2%). Serotype III/ST17 was predominant in iGBS (12/41, 29.3%), and all 17 ST2 strains were noninvasive. The distribution of most of the investigated virulence factors was not significantly related to invasiveness; noteworthily, most of the serotype III/ST17 iGBS carried pilus island (PI) 2b (10/12, 83.3%), and the prevalence of fbsB was significantly low compared with noninvasive GBS isolates (P = 0.004). Characteristically, the combination of bca(+)-cspA(+)-pavA(+)-fbsB(-)-rib(+)-bac(-) was predominant in iGBS (24.4%, 10/41).ConclusionsSerotype III/ST17 GBS carrying PI-2b was frequently detected in iGBS. There was no significant association between invasiveness and the pattern of virulence factors; however, a specific combination of virulence factors was predominant in iGBS.

The dual M protein systems have diverse biological characteristics, but both contribute to M18-type Group A Streptococcus pathogenicity

M protein is a key surface virulence factor in Group A Streptococcus (GAS), Group C Streptococcus (GCS), and other streptococcal species. GAS encodes M protein using the emm gene, while GCS employs the szm (or sem) gene. In M18-type GAS, dual M protein systems exist, comprising both GAS and GCS M proteins (encoded separately by emm18 and spa18). The spa18 gene in M18-type GAS shares a conserved region highly similar to GCS's szm gene. Our study reveals that spa18 exhibits higher transcription levels than emm18 in M18-type GAS strains. The dual M protein systems defective mutant (Δemm18Δspa18) displays a smooth surface, whereas wild-type and single M protein gene mutants remain rough. M18 and SPA18 proteins possess distinct characteristics, showing varied binding properties and cytotoxicity effects on macrophages (THP-1) and keratinocytes (HaCaT). Both emm18 and spa18 genes contribute to the skin pathogenicity of M18-type GAS. Transcriptome analysis suggests the potential involvement of the mga gene in spa18 transcription regulation, while SpyM18_2047 appears to be specific to spa18 regulation. In summary, this research offers a crucial understanding of the biological characteristics of dual M protein systems in M18-type GAS, highlighting their contributions to virulence and transcriptional regulation.

Application of metagenomic next-generation sequencing in the diagnosis of urinary tract infection in patients undergoing cutaneous ureterostomy.

Urinary tract infection (UTI) is an inflammatory response of the urothelium to bacterial invasion and is a common complication in patients with cutaneous ureterostomy (CU). For such patients, accurate and efficient identification of pathogens remains a challenge. The aim of this study included exploring utility of metagenomic next-generation sequencing (mNGS) in assisting microbiological diagnosis of UTI among patients undergoing CU, identifying promising cytokine or microorganism biomarkers, revealing microbiome diversity change and compare virulence factors (VFs) and antibiotic resistance genes (ARGs) after infection. We performed a case-control study of 50 consecutive CU patients from December 2020 to January 2021. According to the clinical diagnostic criteria, samples were divided into infected group and uninfected group and difference of urine culture, cytokines, microorganism, ARGs and VFs were compared between the two groups. Inflammatory responses were more serious in infected group, as evidenced by a significant increase in IFN-α (p=0.031), IL-1β (0.023) and IL-6 (p=0.018). Clinical culture shows that there is higher positive rate in infected group for most clinical pathogens like Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida auris etc. and the top three pathogens with positive frequencies were E. coli, K. pneumoniae, and Enterococcus faecalis. Benchmarking clinical culture, the total sensitivity is 91.4% and specificity is 76.3% for mNGS. As for mNGS, there was no significant difference in microbiome α- diversity between infected and uninfected group. Three species biomarkers including Citrobacter freundii, Klebsiella oxytoca, and Enterobacter cloacae are enriched in infected group based on Lefse. E. cloacae were significantly correlated with IL-6 and IL-10. K. oxytoca were significantly correlated with IL-1β. Besides, the unweighted gene number and weighted gene abundance of VFs or ARGs are significantly higher in infected group. Notablely, ARGs belonging to fluoroquinolones, betalatmas, fosfomycin, phenicol, phenolic compound abundance is significantly higher in infected group which may have bad effect on clinical treatment for patients. mNGS, along with urine culture, will provide comprehensive and efficient reference for the diagnosis of UTI in patients with CU and allow us to monitor microbial changes in urine of these patients. Moreover, cytokines (IL-6, IL-1β, and IFN-a) or microorganisms like C. freundii, K. oxytoca or E. cloacae are promising biomarkers for building effective UTI diagnostic model of patients with CU and seriously the VFs and ARGs abundance increase in infected group may play bad effect on clinical treatment.

Role of Virulence Factors of Trypanosomatids in the Insect Vector and Putative Genetic Events Involved in Surface Protein Diversity.

Trypanosomatids are flagellate protozoans that can infect several invertebrate and vertebrate hosts, including insects and humans. The three most studied species are the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. which are the causative agents of Human African Trypanosomiasis (HAT), Chagas disease and different clinical forms of leishmaniasis, respectively. These parasites possess complex dixenous life cycles, with zoonotic and anthroponotic stages, and are transmitted by hematophagous insects. To colonize this myriad of hosts, they developed mechanisms, mediated by virulence factors, to infect, propagate and survive in different environments. In insects, surface proteins play roles in parasite attachment and survival in the insect gut, whilst in the mammalian host, the parasites have a whole group of proteins and mechanisms that aid them invading the host cells and evading its immune system components. Many studies have been done on the impact of these molecules in the vertebrate host, however it is also essential to notice the importance of these virulence factors in the insect vector during the parasite life cycle. When inside the insect, the parasites, like in humans, also need to survive defense mechanisms components that can inhibit parasite colonization or survival, e.g., midgut peritrophic membrane barrier, digestive enzymes, evasion of excretion alongside the digested blood meal, anatomic structures and physiological mechanisms of the anterior gut. This protection inside the insect is often implemented by the same group of virulence factors that perform roles of immune evasion in the mammalian host with just a few exceptions, in which a specific protein is expressed specifically for the insect vector form of the parasite. This review aims to discuss the roles of the virulence molecules in the insect vectors, showing the differences and similarities of modes of action of the same group of molecules in insect and humans, exclusive insect molecules and discuss possible genetic events that may have generated this protein diversity.

The Role of Candida albicans Virulence Factors in the Formation of Multispecies Biofilms With Bacterial Periodontal Pathogens.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the “red complex” species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic “moonlighting” proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Molecular determination, serotyping, antibiotic profile and virulence factors of group B Streptococcus isolated from invasive patients at Arabcare Hospital Laboratory, Palestine

BackgroundStreptococcus agalactiae (group B Streptococcus) is beta-hemolytic, catalase negative, gram-positive cocci, recognized as main bacterial pathogen causing infections in newborns, infants, adults, and elderly people around the world. The aim of this study is to investigate group B Streptococcus samples recovered from invasive patients and determine serotype, virulent genes, and antibiotic-resistant profile of Streptococcus agalactiae in Palestine. MethodsA total of 95 group B Streptococcus strains were isolated from neonates, infants, pregnant and non-pregnant women and males at Arabcare Hospital Laboratory, Palestine, between the period of June 2018 and September 2020. Species identification was carried out through cultivation and conventional biochemical tests. A conventional Polymerase Chain Reaction (cPCR) was used to determine the 5 serotypes and virulent genes of the Streptococcus agalactiae strains. The antibiotic resistance test of group B Streptococcus was evaluated using Kirby-Bauer disk susceptibility. Sequencing and BLAST analysis were used to determine the relationship of the isolates in this study to worldwide isolates. ResultsSerotype III (35%) was the major group B Streptococcus strains serotype causing invasive infections in neonates, infants, pregnant and nonpregnant women, and males, followed by serotypes V (19%), Ia, and II (15%), Ib (6%), respectively. All our isolates encoding for surface protein virulent factors, including a highly virulent gene (HvgA) were mostly found in strains isolated from pregnant women (12%). These group B Streptococcus strains exhibited a high rate of resistance to clindamycin (26%). The overall percentage of levofloxacin resistance was 11%, while vancomycin and ampicillin showed higher resistance, at 14.7 and 16% respectively. In addition, the phylogenetic relationship dendrogram illustrates that Streptococcus agalactiae isolated from an invasive patient (newborn) in Palestine was similar to strains found in China and Japan. ConclusionsThe outcomes of this study demonstrate that resistant group B Streptococcus strains are common in Palestine, therefore, evidence-based infection prevention and antibiotic stewardship efforts are necessary.

Playing With Fire: Proinflammatory Virulence Mechanisms of Group A Streptococcus.

Group A Streptococcus is an obligate human pathogen that is a major cause of infectious morbidity and mortality. It has a natural tropism for the oropharynx and skin, where it causes infections with excessive inflammation due to its expression of proinflammatory toxins and other virulence factors. Inflammation directly contributes to the severity of invasive infections, toxic shock syndrome, and the induction of severe post-infection autoimmune disease caused by autoreactive antibodies. This review discusses what is known about how the virulence factors of Group A Streptococcus induce inflammation and how this inflammation can promote disease. Understanding of streptococcal pathogenesis and the role of hyper-immune activation during infection may provide new therapeutic targets to treat the often-fatal outcome of severe disease.

Diclofenac mitigates virulence of multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that has the ability to cause a wide range of diseases including superficial infection and severe invasive life threatening infections. The pathogenicity of S. aureus is mediated by a group of virulence factors that mediate the colonization and penetration. The antibiotic resistance of S. aureus has evolved due to the abuse of antibiotics rendering the cure of infection very difficult especially with the shortage in new antibiotic production. To combat this shortage, repurposing of FDA-approved drugs against the virulence factors is a new strategy. The analgesic drug Diclofenac was found to have anti-virulence activity against Pseudomonas aeruginosa and Proteus mirabilis. This study aimed to demonstrate the anti-virulence effect of diclofenac against clinical MRSA isolates phenotypically and genotypically using qRT-PCR. In this study, diclofenac showed significant reduction in biofilm formation when compared to controls, the inhibition ranged between 22.67% and 70%. Also, remarkable inhibition of hemolysin activity was found (5.4-66.34%). Additionally, diclofenac has inhibitory activity against the staphyloxanthin production (8-57.2%). The results were confirmed by qRT-PCR that showed significant down-regulation of tested virulence genes. The down-regulation ranged from 43 to 64.05% for SarA, 36.85-64.75% for AgrA, 50-63.2% for hla, 38.55-60.35% for FnbA, 46.75-61.05% for IcaA, 27.55-64% for SigB and 51.05-72.8% for CrtM. In conclusion, diclofenac can be used in combination with antibiotics as anti-virulence agent against MDR-MRSA which will enhance the ability of immune system to eradicate infection.

Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses.

Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins’ genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.

A Conserved Streptococcal Virulence Regulator Controls the Expression of a Distinct Class of M-Like Proteins.

Streptococcus equi subspecies zooepidemicus (SEZ) are group C streptococci that are important pathogens of economically valuable animals such as horses and pigs. Here, we found that many SEZ isolates bind to a monoclonal antibody that recognizes poly-N-acetylglucosamine (PNAG), a polymer that is found as a surface capsule-like structure on diverse microbes. A fluorescence-activated cell sorting-based transposon insertion sequencing (Tn-seq) screen, coupled with whole-genome sequencing, was used to search for genes for PNAG biosynthesis. Surprisingly, mutations in a gene encoding an M-like protein, szM, and the adjacent transcription factor, designated sezV, rendered strains PNAG negative. SezV was required for szM expression and transcriptome analysis showed that SezV has a small regulon. SEZ strains with inactivating mutations in either sezV or szM were highly attenuated in a mouse model of infection. Comparative genomic analyses revealed that linked sezV and szM homologues are present in all SEZ, S. equi subspecies equi (SEE), and M18 group A streptococcal (GAS) genomes in the database, but not in other streptococci. The antibody to PNAG bound to a wide range of SEZ, SEE, and M18 GAS strains. Immunochemical studies suggest that the SzM protein may be decorated with a PNAG-like oligosaccharide although an intact oligosaccharide substituent could not be isolated. Collectively, our findings suggest that the szM and sezV loci define a subtype of virulent streptococci and that an antibody to PNAG may have therapeutic applications in animal and human diseases caused by streptococci bearing SzM-like proteins.IMPORTANCE M proteins are surface-anchored virulence factors in group A streptococci, human pathogens. Here, we identified an M-like protein, SzM, and its positive regulator, SezV, in Streptococcus equi subspecies zooepidemicus (SEZ), an important group of pathogens for domesticated animals, including horses and pigs. SzM and SezV homologues were found in the genomes of all SEZ and S. equi subspecies equi and M18 group A streptococcal strains analyzed but not in other streptococci. Mutant SEZ strains lacking either sezV or szM were highly attenuated in a mouse model of infection. Collectively, our findings suggest that SezV-related regulators and the linked SzM family of M-like proteins define a new subset of virulent streptococci.

Comparative genome analysis of 15 clinical Shigella flexneri strains regarding virulence and antibiotic resistance.

Shigellosis is the major cause of dysentery globally. It is mainly attributed to two Shigella species, Shigella sonnei and Shigella flexneri, which leads to approximately 165 million infections and 1.1 million deaths each year. Rapid increase and widening of spectrum in antibiotics resistance make Shigella hard to be adequately controlled through existing prevention and treatment measures. It has also been observed that enhanced virulence and advent of antibiotic resistance (AR) could arise almost simultaneously. However, genetic linkages between the two factors are missing or largely ignored, which hinders experimental verification of the relationship. In this study, we sequenced 15 clinically isolated S. flexneri strains. Genome assembly, annotation and comparison were performed through routine pipelines. Differential resistant profiles of all 15 S. flexneri strains to nine antibiotics were experimentally verified. Virulence factors (VFs) belonging to 4 categories and 31 functional groups from the Virulence Factor Database (VFDB) were used to screen all Shigella translated CDSs. Distribution patterns of virulence factors were analysed by correlating with the profiles of bacterial antibiotics resistance. In addition, multi-resistant S. flexneri strains were compared with antibiotic-sensitive strains by focusing on the abundance or scarcity of specific groups of VFs. By doing these, a clear view of the relationships between virulence factors and antibiotics resistance in Shigella could be achieved, which not only provides a set of genetic evidence to support the interactions between VFs and AR but could also be used as a guidance for further verification of the relationships through manipulating specific groups of virulence factors.

Differences in SpeB protease activity among group A streptococci associated with superficial, invasive, and autoimmune disease.

The secreted cysteine proteinase SpeB is an important virulence factor of group A streptococci (GAS), whereby SpeB activity varies widely among strains. To establish the degree to which SpeB activity correlates with disease, GAS organisms were recovered from patients with pharyngitis, impetigo, invasive disease or acute rheumatic fever (ARF), and selected for analysis using rigorous sampling criteria; >300 GAS isolates were tested for SpeB activity by casein digestion assays, and each GAS isolate was scored as a SpeB-producer or non-producer. Highly significant statistical differences (p < 0.01) in SpeB production are observed between GAS recovered from patients with ARF (41.5% SpeB-non-producers) compared to pharyngitis (20.5%), invasive disease (16.7%), and impetigo (5.5%). SpeB activity differences between pharyngitis and impetigo isolates are also significant, whereas pharyngitis versus invasive isolates show no significant difference. The disproportionately greater number of SpeB-non-producers among ARF-associated isolates may indicate an altered transcriptional program for many rheumatogenic strains and/or a protective role for SpeB in GAS-triggered autoimmunity.

The virulence factors of group A streptococcus strains isolated from invasive and non-invasive infections in Polish and German centres, 2009-2011.

Streptococcus pyogenes (GAS) is one of the major human pathogenic bacteria that cause a wide range of diseases. Currently, increased incidence of streptococcal invasive infections is observed worldwide. In this study, we focused on the prevalence of genes encoding superantigens and type M proteins in the population of GAS strains from invasive versus non-invasive infections. We tested 253 GAS strains: 48 strains from patients with invasive infections (18 from wound/deep skin localization, 30 from women in labour) and 205 strains from non-invasive forms (147 from common infections of the upper respiratory, 49 from the vagina of females with genital tract infections and 9 from non-invasive wound and superficial skin infections). Significant differences were found in the occurrence of genes: speG, speI, speJ and smeZ, which were more common in GAS isolated from invasive than from non-invasive strains; speJ and smeZ occurred more frequently in strains from invasive perinatal infections versus strains from women without symptoms of invasive infection; speH and speI in strains from invasive skin/wound infection versus strains isolated from non-invasive wound and superficial skin infections. Emm types 1 and 12 predominated in the group of strains isolated from superficial infections and type 28 in those from puerperal fever. Occurrence of genes encoding virulence factors is common in genomic DNA of most of S. pyogenes, regardless whether these streptococcal infections are invasive or non-invasive. On the other hand, it appears that strains with speG, speI, speJ and smeZ genes may have a particular potential for virulence.

Determination of Enzymatic Antioxidant in Iraqi Patients with Chronic Gastritis

Infection of the gastric mucosa with Helicobacter pylori is strongly associated with chronic gastritis, peptic ulcer and gastric cancer. Helicobacter pylori virulence factors include a variety of proteins that are involved in its pathogenesis, such as VacA and CagA. Another group of virulence factors is clearly important for colonization of H.pylori in the gastric mucosa. These include urease, motility factors (flagellin), and Superoxide dismutase (SOD). Because of this organism's microaerophilic nature and the increased levels of reactive oxygen in the infected host, we expect that other factors involved in the response to oxidative stress are likely to be required for virulence. Superoxide dismutase is a nearly ubiquitous enzyme among organisms that are exposed to toxic environments. In this study, we measured the SOD in serum of 80 patients complain from chronic gastritis and infected with H.pylori. 37 patients infected with H.pylori have the CagA gene, and 13 patients are not and also measured the SOD in 30 control groups that not infected with H.pylori. Serum level of SOD was significantly (p&lt;0.05) higher in patients with chronic gastritis compared to controls. Also significantly higher (p&lt;0.001) in patients with chronic gastritis infected with H.pylori positive CagA than patients infected with H.pylori negative CagA.&#x0D; Key words: chronic gastritis, H.pylori, CagA, SOD&#x0D; الخلاصــة

Impetigo - review.

Impetigo is a common cutaneous infection that is especially prevalent in children. Historically, impetigo is caused by either group A β-hemolytic streptococci or Staphylococcus aureus. Currently, the most frequently isolated pathogen is S. aureus. This article discusses the microbiologic and virulence factors of group A β-hemolytic streptococci and Staphylococcus aureus, clinical characteristics, complications, as well as the approach to diagnosis and management of impetigo. Topical agents for impetigo therapy are reviewed.

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.

Novel inhibitors of bacterial virulence: Development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.

Determining Antibody-Binding Site of Streptococcal Pyrogenic Exotoxin B to Protect Mice from Group A Streptococcus Infection

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. SPE B binds and cleaves antibody isotypes and further impairs the immune system by inhibiting complement activation. In this study, we examined the antibody-binding site of SPE B and used it to block SPE B actions during GAS infection. We constructed different segments of the spe B gene and induced them to express different recombinant fragments of SPE B. Using an enzyme-linked immunosorbent assay (ELISA), we found that residues 345–398 of the C-terminal domain of SPE B (rSPE B345–398), but not the N-terminal domain, was the major binding site for antibody isotypes. Using a competitive ELISA, we also found that rSPE B345–398 bound to the Fc portion of IgG. The in vitro functional assays indicate that rSPE B345–398 not only interfered with cleavage of antibody isotypes but also interfered with SPE B-induced inhibition of complement activation. Immunization of BALB/c mice using rSPE B345–398 was able to induce production of a high titer of anti-rSPE B345–398 antibodies and efficiently protected mice from GAS-induced death. These findings suggest that SPE B uses its C-terminal domain to bind the Fc portion of IgG and that immunization of mice with this binding domain (rSPE B345–398) could protect mice from GAS infection.

Knowing is Half the Battle: Targeting Virulence Factors of Group A Streptococcus for Vaccine and Therapeutics

Group A Streptococcus (GAS) is a leading human pathogen that causes a multitude of diseases from pharyngitis, and impetigo, to more severe outcomes such as rheumatoid arthritis and necrotizing fasciitis. GAS remains a global burden as currently no vaccine exists that is completely effective. In this review we highlight recent studies on the virulence of GAS and present several approaches that have extended those findings into aims at combating GAS disease. These and other studies such as recent genome-wide efforts into host-pathogen relationships of GAS disease will likely reveal new targets of intervention. Given the recent rise in GAS strains that have acquired resistance to several types of antibiotics, it is crucial that we continue to increase our knowledge of the mechanisms underlying GAS disease.

Molecular characteristics of pharyngeal and invasive emm3 Streptococcus pyogenes strains from Norway, 1988–2003

A major virulence factor of group A streptococci (GAS) is the M protein. Strains with the M3 type are more often associated with necrotizing fasciitis (NF) and streptococcal toxic shock syndrome, and have a higher case fatality rate than strains of other M types. To better understand the epidemiology of M3 GAS strains in Norway, we analyzed 59 invasive and 69 pharyngeal isolates with respect to prophage content, allelic variation in emm3, mtsR encoding the metal transporter of Streptococcus repressor (mtsR), and sclB coding for streptococcal collagen-like protein B. The Norwegian emm3 strains were very homogeneous, mainly harboring the emm allele 3.1 and prophage profile PhiG3.01. Other prophage profiles were transient. The mutation in mtsR known to truncate the protein and result in decreased capacity to cause NF was not found in our isolates. The sclB gene usually harbored five or eight contiguous repeats of a CAAAA pentanucleotide sequence and a highly modular and variable collagen structural motif (CSM) region with 9 and 12 amino acid M3-specific conserved motif repeats distributed across the entire CSM region. Strains with 5 CAAAA repeats emerged in 1993 and these strains were associated with the increase in invasive M3 cases in the period 1993-2003.