Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae

The ability of pneumococci to take up naked DNA from the environment and permanently incorporate the DNA into their genome by recombination has been exploited as a valuable research tool for 80 years. From being viewed as a marginal phenomenon, it has become increasingly clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity, and that it has the potential to cause severe problems for future treatment of pneumococcal disease. This process constitutes a highly efficient mechanism for spreading β-lactam resistance determinants between streptococcal strains and species, and also threatens to undermine the effect of pneumococcal vaccines. Fortunately, great progress has been made during recent decades to elucidate the mechanism behind natural transformation at a molecular level. Increased insight into these matters will be important for future development of therapeutic strategies and countermeasures aimed at reducing the spread of hazardous traits. In this review, we focus on recent developments in our understanding of competence regulation, DNA acquisition and the role of natural transformation in the dissemination of virulence and β-lactam resistance determinants.     

Mobile Genetic Element-Encoded Cytolysin Connects Virulence to Methicillin Resistance in MRSA

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.     

MRSA epidemic linked to a quickly spreading colonization and virulence determinant

The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) infection are poorly understood(1). Although a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer(2), there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare(3,4) mobile genetic element–encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, substantially enhancing nasal colonization, lung disease and abscess formation and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type 239 (ST239) to invasive clones belonging to other sequence types. Our study identifies sasX as a quickly spreading crucial determinant of MRSA pathogenic success and a promising target for therapeutic interference. Our results provide proof of principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.     

The role of transformation in the variability of the Neisseria gonorrhoeae cell surface

This review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation-mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin-encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus-specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation-mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.     

鸭疫里默氏杆菌自然转化的发现及机制研究进展

自然转化(natural transformation)是微生物水平基因转移的一种重要机制,其在遗传多样性的产生或修复DNA损伤等方面发挥着重要作用,并且与耐药基因、毒力因子的扩散息息相关。鸭疫里默氏杆菌(Riemerellaanatipestifer,RA)是威克斯菌科中第一个被发现可以发生自然转化的细菌,本文作者利用此特点建立了多种基因编辑的方法,促进了对其遗传多样性和致病机理的研究进程。通过系统性地研究影响鸭疫里默氏杆菌自然转化的因素,鉴定出了参与该菌自然转化的营养物质;通过筛选转座子插入突变体文库,鉴定出了参与该过程的必需基因;最终,在该菌发现了一种新型的自然转化系统。本文结合其他细菌自然转化研究进展,针对以上研究结果进行综述,以期对该菌的自然转化机制有更深入的理解,也为更进一步探明该菌的耐药和毒力基因获得机制提供参考。     

Disruption of the cyanide hydratase gene in Gloeocercospora sorghi increases its sensitivity to the phytoanticipin cyanide but does not affect its pathogenicity on the cyanogenic plant sorghum

The release of hydrogen cyanide (HCN) from preformed cyanogenic compounds in plants such as sorghum is thought to provide a protective barrier against infection by microorganisms. Gloeocercospora sorghi, a fungal pathogen of sorghum, produces the enzyme cyanide hydratase (CHT) which converts HCN to the less toxic compound formamide. There is considerable prior evidence indicating that this mechanism for detoxifying HCN plays an important role in the pathogenicity of G. sorghi on sorghum. In the present study, the CHT gene was made nonfunctional in G. sorghi through transformation-mediated gene disruption. The transformant lacked CHT activity and no reacting polypeptides were detected with CHT-specific antibodies. This CHT mutant was highly sensitive to HCN, confirming that CHT is an HCN detoxifying mechanism, but it retained virulence on sorghum, causing lesions indistinguishable from those caused by the wild-type strain. This result indicates that G. sorghi does not require CHT for pathogenicity on cyanogenic lines of sorghum and suggests that cyanogenic compounds in plants may serve functions other than providing a mechanism of disease resistance.     

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.     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

病原菌毒力岛研究进展

毒力岛作为基因组岛的一种亚类,是细菌染色体上具有特定结构和功能特征的可移动基因大片段,经基因水平转移（转导、接合或转化）获得,可使细菌基因组进化在短期内发生“量的飞跃”,直接或间接增强细菌的生态适应性,与病原菌的致病性密切相关。毒力岛存在于多种动植物病原细菌中,对于细菌的毒力变异、遗传进化甚至新病原亚种形成有重要意义。简要综述了病原菌毒力岛的研究进展,介绍了毒力岛的结构、功能特征及其在病原菌进化中作用。     

Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae – a review

In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.     

Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes

Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts and hepatocytes. InlF, a member of the internalin multigene family with no known function, was identified as a L. monocytogenes -specific factor mediating increased host cell binding and entry. Conversely, activation of RhoA/ROCK activity resulted in decreased L. monocytogenes adhesion and invasion. Furthermore, virulence of wild-type bacteria during infection of mice was significantly increased upon inhibition of ROCK activity, whereas colonization and virulence of an inlF deletion mutant was not affected, thus supporting a role for InlF as a functional virulence determinant in vivo under specific conditions. In addition, inhibition of ROCK activity in human-derived cells enhanced either bacterial adhesion or adhesion and entry in an InlF-independent manner, further suggesting a host species or cell type-specific role for InlF and that additional bacterial determinants are involved in mediating ROCK-regulated invasion of human cells.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island

Enterococcus faecalis, a leading cause of nosocomial antibiotic resistant infections, frequently possesses a 150 kb pathogenicity island (PAI) that carries virulence determinants. The presence of excisionase and integrase genes, conjugative functions and multiple insertion sequence elements suggests that the PAI, or segments thereof, might be capable of horizontal transfer. In this report, the transfer of the E. faecalis PAI is demonstrated and a mechanism for transfer elucidated. In filter matings, chloramphenicol resistance was observed to transfer from strain MMH594b, a clinical isolate possessing the PAI tagged with a cat marker, to OG1RF (pCGC) with a frequency of 3.2 x 10(-10) per donor. Secondary transfer from primary transconjugant TCRFB1 to strain JH2SS in filter and broth matings occurred with a frequency of 1 and 2 x 10(-1) per donor respectively. Analysis of the transconjugants demonstrated that a 27,744 bp internal PAI segment was capable of excision and circularization in the donor, and is mobilized as a cointegrate with a pTEF1-like plasmid. High-frequency transfer also occurred from TCRFB1 to JH2SS during transient colonization of the mouse gastrointestinal tract. This is the first demonstration of the horizontal transfer of PAI-encoded virulence determinants in E. faecalis and has implications for genome evolution and diversity.     

Signature-tagged mutagenesis in the identification of virulence genes in pathogens

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.     

Experimental adaptation of an RNA virus mimics natural evolution

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.     

Molecular Screening of Enterococcus Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Iron and Pathogenesis of Shigella: Iron Acquisition in the Intracellular Environment

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.