Genomic approaches in Xanthomonas campestris pv. vesicatoria allow fishing for virulence genes

Xanthomonas campestris pv. vesicatoria is an economically important pathogen of pepper and tomato and has been established as a model organism to study bacterial infection strategies. In the last two decades, intensive genetic and molecular analyses led to the isolation of many genes that play a role in the intimate molecular relationship with the host plant. Essential for pathogenicity is a type III protein secretion system, which delivers bacterial effector proteins into the host cell. Currently, the genome of X. campestris pv. vesicatoria is being sequenced. The availability of genomic sequence information will pave the way for the identification of new bacterial virulence factors by bioinformatic approaches. In this article, we will present preliminary data from the genomic sequence analysis and describe recent and novel studies to identify bacterial type III effector genes.     

A functional genomic yeast screen to identify pathogenic bacterial proteins

Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.     

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.     

选择性捕获禽病原性大肠杆菌体内转录序列

采用选择性捕获转录序列(SCOTS)方法鉴定禽病原性大肠杆菌E037株(血清型O78)在感染SPF鸡过程中的转录表达基因。通过总RNA分离、cDNAs合成、PCR扩增和SCOTS对cDNAs选择和致病性特异转录序列的富集,致病性特异的cDNAs被分离鉴定,共获得31个转录序列(命名为aec),其中分别有2、1、4、14、2和8个aec序列与黏附素、LPS的合成、铁的摄取系统、质粒编码基因、噬菌体编码基因和一些其它功能基因相关;从气囊中分离到16个aec序列,心包膜中分离到15个aec序列;有3种与质粒编码基因相关序列在气囊和心包膜中都被分离到。结果显示APEC致病性特异序列包括黏附素、LPS的合成、铁的转运、质粒编码基因、噬菌体编码基因和一些其它功能基因等。通过SCOTS方法建立了一种体内表达致病性特异基因的方法和APEC在自然宿主感染模型中致病性相关基因的表达谱的筛选方法。     

Bacterial virulence as a target for antimicrobial chemotherapy

As bacterial resistance to currently used antibiotics increases, so too must efforts to identify novel agents and strategies for the prevention and treatment of bacterial infection. In the past, antimicrobial drug discovery efforts have focused on eradicating infection by either cidal or static agents, resulting in clearance of the bacterium from the infected host. To this end, drug discovery targets have been those proteins or processes essential for bacterial cell viability. However, inhibition of the interaction between the bacterium and its host may also be a target. During establishment of an infection, pathogenic bacteria use carefully regulated pathways of conditional gene expression to transition from a free-living form to one that must adapt to the host milieu. This transition requires the regulated production of both extracellular and cell-surface molecules, often termed virulence factors. As the biological imperatives of the invading organism change during the course of an infection, the expression of these factors is altered in response to environmental cues. These may be changes in the host environment, for example, pH, metabolites, metal ions, osmolarity, and temperature. Alternatively, effector molecules produced by the bacterium to sense changing cell density can also lead to changes in virulence gene expression. Although the mechanisms of pathogenesis among different bacteria vary, the principles of virulence are generally conserved. Bacterial virulence may therefore offer unique opportunities to inhibit the establishment of infection or alter its course as a method of antimicrobial chemotherapy.     

Measurement of bacterial gene expression in vivo

The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments. We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful. This response involves hundreds of ivi (in vivo-induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display. Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens. The patterns of bacterial gene expression during infection remain to be investigated. Are ivi genes expressed in an organ-specific or cell-type-specific fashion? New approaches are required to answer these questions. The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered. This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.     

Proteolysis and its regulation at the surface of Streptococcus pyogenes

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus pyogenes during infection with this important human pathogen. Streptococcus pyogenes produces proteinases, and host proteinases are produced and released as a result of the infection. Streptococcus pyogenes also recruits host proteinase inhibitors to its surface, suggesting that proteolysis is tightly regulated at the bacterial surface. We propose that the initial phase of a S. pyogenes infection is characterized by inhibition of proteolysis and complement activity at the bacterial surface. This is achieved mainly through binding of host proteinase inhibitors and complement regulatory proteins to bacterial surface proteins. In a later phase of the infection, massive proteolytic activity will release bacterial surface proteins and degrade human tissues, thus facilitating bacterial spread. These proteolytic events are regulated both temporally and spatially, and should influence virulence and the outcome of S. pyogenes infections.     

The type III needle and the damage done

Many Gram-negative pathogens translocate virulence proteins directly into host cells using a type III secretion system. This complex secretion machinery is composed of approximately 25 different proteins that assemble to span both bacterial membranes, and contact the host cell to form a direct channel between the bacterial and host cell cytoplasms. Assembly of the system and efficient secretion of virulence proteins through this apparatus require specific chaperones. Although the machinery is morphologically conserved among all bacteria, the secreted proteins vary widely and are responsible for the range of diseases caused by bacterial pathogens. Recent structures have given insights into important chaperone and effector proteins, as well as revealing the first atomic structures of portions of the secretion machinery itself.     

Biomedicals from a soil bug: expanding scFv production host range

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem. The development of inducible broad host range scFv expression plasmid constructs leads the way to an easy and efficient screening method for the identification of the optimal bacterial expression host.     

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.     

Bioinformatics Annotation of the Hypothetical Proteins Found by Omics Techniques Can Help to Disclose Additional Virulence Factors

The advent of genomics should have facilitated the identification of microbial virulence factors, a key objective for vaccine design. When the bacterial pathogen infects the host it expresses a set of genes, a number of them being virulence factors. Among the genes identified by techniques as microarrays, in vivo expression technology, signature-tagged mutagenesis and differential fluorescence induction there are many related to cellular stress, basal metabolism, etc., which cannot be directly involved in virulence, or at least cannot be considered useful candidates to be deleted for designing a live attenuated vaccine. Among the genes disclosed by these methodologies there are a number of hypothetical or unknown proteins. As they can hide some true virulence factors, we have reannotated all of these hypothetical proteins from several respiratory pathogens by a careful and in-depth analysis of each one. Although some of the re-annotations match with functions that can be related to microbial virulence, the identification of virulence factors remains difficult.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.     

Global proteomic pattern of Tropheryma whipplei: A Whipple's disease bacterium

The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2‐DE coupled with MALDI‐TOF and SDS‐PAGE with nanoLC‐MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N‐terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species‐specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host‐associated WD bacterium with other host‐associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host‐associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.     

Quantitative analysis of Streptococcus pneumoniae TIGR4 response to in vitro iron restriction by 2-D LC ESI MS/MS

Understanding the growth of bacterial pathogens in a micronutrient restricted host environment can identify potential virulence proteins that help overcome this nutritional barrier to productive infection. In this study, we investigated the pneumococcal protein expression response to iron limitation using an in vitro model. We identified S. pneumoniae TIGR4 proteins by 2-D LC ESI MS/MS and determined significant changes in protein expression in response to iron restriction using computer-intensive random resampling methods. Differential protein expression was studied in the context of a S. pneumoniae TIGR4 protein interaction network using Pathway Studio. Our analysis showed that pneumococcal iron restriction response was marked by increased expression of known virulence factors like PsaA. It involved changes in the expression of stress response, and phase variation and biofilm formation proteins. The net effect of changes in all these biological processes could increase the virulence of S. pneumoniae TIGR4 during in vivo infection.     

Infection in a dish: high-throughput analyses of bacterial pathogenesis

Diverse aspects of host-pathogen interactions have been studied using non-mammalian hosts such as Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio for more than 20 years. Over the past two years, the use of these model hosts to dissect bacterial virulence mechanisms has been expanded to include the important human pathogens Vibrio cholerae and Yersinia pestis. Innovative approaches using these alternative hosts have also been developed, enabling the isolation of new antimicrobials through screening large libraries of compounds in a C. elegans Enterococcus faecalis infection model. Host proteins required by Mycobacterium and Listeria during their invasion and intracellular growth have been uncovered using high-throughput dsRNA screens in a Drosophila cell culture system, and immune evasion mechanisms deployed by Pseudomonas aeruginosa during its infection of flies have been identified. Together, these reports further illustrate the potential and relevance of these non-mammalian hosts for modelling many facets of bacterial infection in mammals.     

Playing Dr Jekyll and Mr Hyde: combined mechanisms of phase variation in bacteria

Phase variation is the adaptive process by which bacteria undergo frequent and reversible phenotypic changes resulting from genetic alterations in specific loci of their genomes. This process is crucial for the survival of pathogens and commensals in hostile and ever-changing host environments. Despite important differences in the molecular mechanisms that mediate and regulate phase variation, related strategies have evolved to generate high levels of genetic diversity through complex and combinatorial reshuffling of genetic information. Recent studies, supported by the emergence of global genomic approaches, have revealed that bacterial pathogens often use a combination of different mechanisms to vary the expression of a variety of biological functions, providing new insights into bacterial adaptation and virulence mechanisms. Recent advances in the understanding of the molecular mechanisms of phase variation are reviewed, and differences in these mechanisms outlined.