Extracellular proteinases of Candida species pathogenic yeasts

The increased incidence of severe disseminated infections caused by the opportunistic yeast‐like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens—extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein–kinin system), a dysregulated host proteinase–inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.     

Synergistic findings from microbiological and evolutional analyses of virulence factors among pathogenic streptococcal species

Background

Members of the genus Streptococcus are major constituents of human skin and the mucosal microbiome, among which Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae are extracellular pathogens that occasionally cause life-threatening infectious diseases. Prior to their successful spread into the blood and deeper organs, pathogenic bacteria must first colonize human epithelial cell surfaces and evade host immunity. Streptococcal extracellular proteins play important roles in this colonization, as they directly interact with the host environment.

Molecular pathogenicity of Streptococcus anginosus

Streptococcus anginosus and the closely related species Streptococcus constellatus and Streptococcus intermedius, are primarily commensals of the mucosa. The true pathogenic potential of this group has been under‐recognized for a long time because of difficulties in correct species identification as well as the commensal nature of these species. In recent years, streptococci of the S. anginosus group have been increasingly found as relevant microbial pathogens in abscesses and blood cultures and they play a pathogenic role in cystic fibrosis. Several international studies have shown a surprisingly high frequency of infections caused by the S. anginosus group. Recent studies and a genome‐wide comparative analysis suggested the presence of multiple putative virulence factors that are well‐known from other streptococcal species. However, very little is known about the molecular basis of pathogenicity in these bacteria. This review summarizes our current knowledge of pathogenicity factors and their regulation in S. anginosus.     

Towards defining the outer membrane proteome of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram‐negative anaerobic pathogen found in subgingival plaque associated with progressive periodontitis. Proteins associated with the outer membrane (OM) of Gram‐negative pathogens are particularly important for understanding virulence and for developing vaccines. The aim of this study was to establish a reliable list of outer membrane associated proteins (Omps) for this organism. Starting with a list of 99 experimentally determined Omps, several bioinformatics tools were used to predict a further 52 proteins, leading to a predicted OM proteome of 151 proteins. The tools used included databases of protein families, prediction of OM β‐barrels and structural homology. The list includes 33 T9SS cargo proteins, 43 lipoproteins and 66 OM β‐barrel proteins with some overlap between categories. The proteins are discussed both in these structural categories as well as their various functions in OM biogenesis, nutrient acquisition, protein secretion, adhesion and efflux. Proteins that were previously shown to be part of large complexes are highlighted and cross reference is provided to a previous major study of protein localization in P. gingivalis. Finally, proteins were also scored according to their level of conservation within the Bacteroidales taxon. Low scores were shown to correlate with virulence factors and may be predictive of novel virulence factors.     

Pneumococcal surface proteins: when the whole is greater than the sum of its parts

Surface-exposed proteins of pathogenic bacteria are considered as potential virulence factors through their direct contribution to host-pathogen interactions. Four families of surface proteins decorate the cell surface of the human pathogen Streptococcus pneumoniae. Besides lipoproteins and LPXTG proteins, also present in other gram-positive bacteria, the pneumococcus presents the choline-binding protein (CBP) family and the non-classical surface proteins (NCSPs). The CBPs present specific structural features that allow their anchorage to the cell envelope through non-covalent interaction with choline residues of lipoteichoic acid and teichoic acid. NCSP is an umbrella term for less characterized proteins displaying moonlighting functions on the pneumococcal surface that lack a leader peptide and membrane-anchor motif. Considering the unceasing evolution of microbial species under the selective pressure of antibiotic use, detailed understanding of the interaction between pathogen and the host cells is required for the development of novel therapeutic strategies to combat pneumococcal infections. This article reviews recent progress in the investigation of the three-dimensional structures of surface-exposed pneumococcal proteins. The modular nature of some of them produces a great versatility and sophistication of the virulence functions that, in most cases, cannot be deduced by the structural analysis of the isolated modules.     

Polyamines in the virulence of bacterial pathogens of respiratory tract

Polyamines are positively charged hydrocarbons that are essential for the growth and cellular maintenance in prokaryotes and eukaryotes. Polyamines have been demonstrated to play a role in bacterial pathogenicity and biofilm formation. However, the role of extracellular polyamines as a signaling molecule in the regulation of virulence is not investigated in detail. The bacterial pathogens residing in the respiratory tract remain asymptomatic for an extended period; however, the factors that lead to symptomatic behavior are poorly understood. Further investigation to understand the relation between the host‐secreted factors and virulence of pathogenic bacteria in the respiratory tract may provide insights into the pathogenesis of respiratory tract infections. Polyamines produced within the bacterial cell are generally sequestered. Therefore, the pool of extracellular polyamines formed by secretion of the commensals and the host may be one of the signaling molecules that might contribute toward the alterations in the expression of virulence factors in bacterial pathogens. Besides, convergent mechanisms of polyamine biosynthesis do exist across the border of species and genus level. Also, several novel polyamine transporters in the host and bacteria remain yet to be identified. The review focuses on the role of polyamines in the expression of virulence phenotypes and biofilm formation of the respiratory tract pathogens.     

Streptococcus mutans copper chaperone,CopZ, is critical for biofilm formation and competitiveness

The oral cavity is a dynamic environment characterized by hundreds of bacterial species, saliva, and an influx of nutrients and metal ions such as copper. Although there is a physiologic level of copper in the saliva, the oral cavity is often challenged with an influx of copper ions. At high concentrations copper is toxic and must therefore be strictly regulated by pathogens for them to persist and cause disease. The cariogenic pathogen Streptococcus mutans manages excess copper using the copYAZ operon that encodes a negative DNA‐binding repressor (CopY), the P1‐ATPase copper exporter (CopA), and the copper chaperone (CopZ). These hypothetical roles of the copYAZ operon in regulation and copper transport to receptors led us to investigate their contribution to S. mutans virulence. Mutants defective in the copper chaperone CopZ, but not CopY or CopA, were impaired in biofilm formation and competitiveness against commensal streptococci. Characterization of the CopZ mutant biofilm revealed a decreased secretion of glucosyltransferases and reduced expression of mutacin genes. These data suggest that the function of copZ on biofilm and competitiveness is independent of copper resistance and CopZ is a global regulator for biofilm and other virulence factors. Further characterization of CopZ may lead to the identification of new biofilm pathways.     

Analysis of the protease and adhesin domains of the PrpRI of Porphyromonas gingivalis

The production of extracellular proteolytic enzymes is a widely used strategy by human parasites including bacteria, protozoa and helminths in order to ensure survival in the colonized host. The potential benefits to the organism arise through modifications to the external environment of the cell and include the release of essential nutrients, the disablement/deregulation of the host defences and the exposure of previously shielded substrata as new sites for colonization. Damage to the host may arise through direct proteolysis of structural proteins, deregulation of the inflammatory response or the compromising of the local host defences below the threshold necessary for effective defence. In order to examine these interactions and how they may be regulated in the periodontal diseases, we are examining the properties of proteases of the oral anaerobe Porphyromonas gingivalis with specificity for arginyl peptide bonds (ArgI, ArgIA and ArgIB): a family of enzymes which has been shown to exert effects on a variety of host proteins with roles in the control of inflammation and tissue homeostasis. Analysis of the gene for ArgI (protease polyprotein for ArgI - prpRl) together with structural and immunochemical studies of these 3 interrelated forms indicates that they may be regarded as critical determinants in multiple aspects of the life cycle of the organism via both proteolysis and binding processes. Together with the highly conserved nature of the gene, the data suggest that the PrpRI of P. gingivalis is an essential colonization determinant which may play an important role in the periodontal disease process.     

Learning the ABC of oral fungal drug resistance

ATP‐binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy‐dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high‐level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.     

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net‐like fibers comprising DNA and antimicrobial components such as histones, LL‐37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram‐negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA‐ and nucD‐encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg²⁺ and Ca²⁺ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.     

细菌非编码小RNA对细菌毒力的调控作用

在病原菌与宿主长期的交互作用过程中,病原菌可通过调节相关基因的表达响应宿主微环境的变化,以适应宿主内环境并在宿主体内生存。过去认为,细菌的基因表达调控主要发生在转录水平。近年发现,细菌非编码小RNA（sRNA）在调控细菌致病机制方面发挥着重要作用。细菌sRNA是一类长度在50~500个核苷酸之间的非编码RNA,病原菌可感受宿主微环境的变化,通过sRNA调控自身毒力相关基因的表达,促进致病菌在宿主内的生存能力,利于致病菌对宿主的侵袭及致病。相对于各种转录因子,sRNA介导的基因表达调控可使细菌更快速、灵敏地对外界环境变化作出应答。目前已发现多种与细菌毒力及致病性相关的sRNA,可在转录后水平精细调节细菌毒力及其致病机制。本文就sRNA调控细菌毒力的分子机制及其在常见病原菌致病过程中的作用进行综述。     

Virulence factors of the oral spirochete Treponema denticola

There is compelling evidence that treponemes are involved in the etiology of several chronic diseases, including chronic periodontitis as well as other forms of periodontal disease. There are interesting parallels with other chronic diseases caused by treponemes that may indicate similar virulence characteristics. Chronic periodontitis is a polymicrobial disease, and recent animal studies indicate that co-infection of Treponema denticola with other periodontal pathogens can enhance alveolar bone resorption. The bacterium has a suite of molecular determinants that could enable it to cause tissue damage and subvert the host immune response. In addition to this, it has several non-classic virulence determinants that enable it to interact with other pathogenic bacteria and the host in ways that are likely to promote disease progression. Recent advances, especially in molecular-based methodologies, have greatly improved our knowledge of this bacterium and its role in disease.     

Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology

Recent advancements in the periodontal research field are consistent with a new model of pathogenesis according to which periodontitis is initiated by a synergistic and dysbiotic microbial community rather than by select ‘periopathogens’, such as the ‘red complex’. In this polymicrobial synergy, different members or specific gene combinations within the community fulfill distinct roles that converge to shape and stabilize a disease‐provoking microbiota. One of the core requirements for a potentially pathogenic community to arise involves the capacity of certain species, termed ‘keystone pathogens’, to modulate the host response in ways that impair immune surveillance and tip the balance from homeostasis to dysbiosis. Keystone pathogens also elevate the virulence of the entire microbial community through interactive communication with accessory pathogens. Other important core functions for pathogenicity require the expression of diverse molecules (e.g. appropriate adhesins, cognate receptors, proteolytic enzymes and proinflammatory surface structures/ligands), which in combination act as community virulence factors to nutritionally sustain a heterotypic, compatible and proinflammatory microbial community that elicits a non‐resolving and tissue‐destructive host response. On the basis of the fundamental concepts underlying this model of periodontal pathogenesis, that is, p olymicrobial s ynergy and d ysbiosis, we term it the PSD model.     

Por Secretion System of Porphyromonas gingivalis

The virulence factors of pathogenic bacteria are major secretory proteins that are directly linked to their pathogenicity. These secretory proteins are translocated across the membranes of bacterial cells by translocase nanomachines, which consists of integral membrane proteins. The periodontal pathogen, Porphyromonas gingivalis, secretes trypsin-like proteases (gingipains) either as a large complex on the bacterial cell surface or into the extracellular milieu. Gingipains are important virulence factors, because they degrade host proteins. They are responsible for the processing/maturation of other P. gingivalis virulence factors. At least six types of translocase nanomachines have been found in Gram-negative bacteria ; however, P. gingivalis does not have genes homologous to those coding these secretion systems in the bacterial genome and not much is known about the mechanism of gingipain secretion. In this study, the proteins responsible for gingipain secretion, i.e., PorK, PorL, PorM, PorN, and PorW, were identified by comparative genome analysis and genetic experiments. We named the gingipain secretion system the Por secretion system (PorSS). Genes encoding PorSS proteins are conserved among a group of bacteria including periodontal pathogens such as Tannellera forsythia and Prevotella intermedia in the phylum Bacteroidetes. In addition, homologous genes are involved in gliding motility and chitinase secretion in Flavobacterium johnsoniae, another member of the phylum Bacteroidetes. Two other genes, porX and porY, encoding the regulatory factors of PorSS gene expression were identified at the same time. The expression of the porT, porK, porL, porM, and porN genes was downregulated in PorX- or PorY-defective mutants. PorSS and its regulatory system appear to be associated with the pathogenicity of various bacteria in the phylum Bacteroidetes.     

Activation of the TREM‐1 pathway in human monocytes by periodontal pathogens and oral commensal bacteria

Periodontitis is a highly prevalent disease caused in part by an aberrant host response to the oral multi‐species biofilm. A balance between the oral bacteria and host immunity is essential for oral health. Imbalances in the oral microbiome lead to an uncontrolled host inflammatory response and subsequent periodontal disease (i.e. gingivitis and periodontitis). TREM‐1 is a signaling receptor present on myeloid cells capable of acting synergistically with other pattern recognition receptors leading to amplification of inflammatory responses. The aim of this study was to investigate the activation of the TREM‐1 pathway in the human monocyte‐like cell line THP‐1 exposed to both oral pathogens and commensals. The relative expression of the genes encoding TREM‐1 and its adapter protein DAP12 were determined by quantitative real‐time polymerase chain reaction. The surface expression of TREM‐1 was determined by flow cytometry. Soluble TREM‐1 and cytokines were measured by enzyme‐linked immunosorbent assay. The results demonstrate that both commensal and pathogenic oral bacteria activate the TREM‐1 pathway, resulting in a proinflammatory TREM‐1 activity‐dependent increase in proinflammatory cytokine production.     

白假丝酵母菌在义齿性口炎中的相关研究

义齿性口炎是一种发生在可摘义齿佩戴者口腔的常见病,其主要的特征为与义齿接触的口腔黏膜出现炎症与红斑或组织水肿样病损,发病机制仍不明确.研究表明,白假丝酵母菌与义齿性口炎的发生有着密切的关系.白假丝酵母菌主要通过其分泌的侵袭酶与黏附素及菌丝相的形成作为毒力因子,对宿主细胞进行感染.这一系列的毒力作用大多由某一种或几种分子通路来实现调控.本文就白假丝酵母菌在义齿性口炎中相关毒力因子的产生与作用,其分子通路及宿主细胞的反应等几个方面进行综述.     

Type IX secretion system is pivotal for expression of gingipain‐associated virulence of Porphyromonas gingivalis

Porphyromonas gingivalis, a keystone pathogen in periodontitis, secretes an array of virulence factors including gingipains via the type IX secretion system (T9SS). Inactivation of any component of the T9SS leads to the accumulation of secreted proteins in unprocessed and, in the case of progingipains, inactive forms in the periplasm. To cast light on the paradox that active gingipains are essential for P. gingivalis fitness in vivo but a functional T9SS is not (Frontiers in Cellular and Infection Microbiology, 2017, 7:378), we have compared virulence of wild‐type P. gingivalis W83 and the gingipain‐null strain with isogenic mutants deficient in individual T9SS components. Using an in vivo subcutaneous chamber mouse model of infection, gingipain‐null strain secretion mutants showed no virulence, but their pathogenic potential was reconstituted by coinfection with a low number of the parental strain. Apparently the same mechanism compensated fitness of mutants lacking functional T9SS the transposon library. In contrast to the parental strain, all mutants elicited significantly lower but an effective inflammatory immune response, which cleared infection and prevented systemic dissemination of P. gingivalis to organs. There were no significant differences in immune responses to different secretion mutants, which were generally more stimulatory than the gingipain‐null strain. Together, these results indicate that functional T9SS is essential for P. gingivalis virulence apparently through delivery of active gingipains to the bacterial surface. Therefore, T9SS is a legitimate target for drug development to treat periodontitis.     

Sialic acid, periodontal pathogens and Tannerella forsythia: stick around and enjoy the feast!

Periodontal pathogens, like any other human commensal or pathogenic bacterium, must possess both the ability to acquire the necessary growth factors and the means to adhere to surfaces or reside and survive in their environmental niche. Recent evidence has suggested that sialic acid containing host molecules may provide both of these requirements in vivo for several periodontal pathogens but most notably for the red complex organism Tannerella forsythia. Several other periodontal pathogens also possess sialic acid scavenging enzymes - sialidases, which can also expose adhesive epitopes, but might also act as adhesins in their own right. In addition, recent experimental work coupled with the release of several genome sequences has revealed that periodontal bacteria have a range of sialic acid uptake and utilization systems while others may also use sialic acid as a cloaking device on their surface to mimic host and avoid immune recognition. This review will focus on these systems in a range of periodontal bacteria with a focus on Ta. forsythia.