Aaron P. White

Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

ABSTRACT Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.

From Exit to Entry: Long-term Survival and Transmission of Salmonella

Salmonella spp. are a leading cause of human infectious disease worldwide and pose a serious health concern. While we have an improving understanding of pathogenesis and the host-pathogen interactions underlying the infection process, comparatively little is known about the survival of pathogenic Salmonella outside their hosts. This review focuses on three areas: (1) in vitro evidence that Salmonella spp. can survive for long periods of time under harsh conditions; (2) observations and conclusions about Salmonella persistence obtained from human outbreaks; and (3) new information revealed by genomic- and population-based studies of Salmonella and related enteric pathogens. We highlight the mechanisms of Salmonella persistence and transmission as an essential part of their lifecycle and a prerequisite for their evolutionary success as human pathogens.

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data

BackgroundAs sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis.ResultsIn this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s.ConclusionsOur results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for comparative analyses with other Salmonella serotypes.

Examining the Link between Biofilm Formation and the Ability of Pathogenic Salmonella Strains to Colonize Multiple Host Species

Salmonella are important pathogens worldwide and a predominant number of human infections are zoonotic in nature. The ability of strains to form biofilms, which is a multicellular behavior characterized by the aggregation of cells, is predicted to be a conserved strategy for increased persistence and survival. It may also contribute to the increasing number of infections caused by ingestion of contaminated fruits and vegetables. There is a correlation between biofilm formation and the ability of strains to colonize and replicate within the intestines of multiple host species. These strains predominantly cause localized gastroenteritis infections in humans. In contrast, there are salmonellae that cause systemic, disseminated infections in a select few host species; these “invasive” strains have a narrowed host range, and most are unable to form biofilms. This includes host-restricted Salmonella serovar Typhi, which are only able to infect humans, and atypical gastroenteritis strains associated with the opportunistic infection of immunocompromised patients. From the perspective of transmission, biofilm formation is advantageous for ensuring pathogen survival in the environment. However, from an infection point of view, biofilm formation may be an anti-virulence trait. We do not know if the capacity to form biofilms prevents a strain from accessing the systemic compartments within the host or if loss of the biofilm phenotype reflects a change in a strain’s interaction with the host. In this review, we examine the connections between biofilm formation, Salmonella disease states, degrees of host adaptation, and how this might relate to different transmission patterns. A better understanding of the dynamic lifecycle of Salmonella will allow us to reduce the burden of livestock and human infections caused by these important pathogens.

The Salmonella Pathogenicity Island-1 and -2 Encoded Type III Secretion Systems

Salmonellae are motile, facultatively anaerobic, Gram-negative rods measuring 0.3-1.5 by 1.02.5 m in size. The genus Salmonella was named for Dr. Daniel Salmon, a veterinary bacteriologist at the United States Department of Agriculture (USDA) (Gast, 2003, Salyers & Whitt, 2002). The Salmonella species are closely related to Escherichia, Yersinia, and Shigella, and contain a circular chromosome approximately 4.7 Mbp in size with an overall GC content of 52% (Marcus, et al., 2000, Salyers & Whitt, 2002, Thomson, et al., 2008). The genus Salmonella lies within the kingdom Eubacteria, class Gammaproteobacteria, order Enterobacteriales, and family Enterobacteriaceae. Salmonella is divided into two species, Salmonella bongori and Salmonella enterica. Within Salmonella enterica there are 6 subspecies: salamae, arizonae, diarizonae, houtenae, indica, and enterica (Tindall, et al., 2005). These subspecies can be further classified into approximately 50 serogroups based on their lipopolysaccharide (LPS) O antigen component (Sabbagh, et al., 2010). Salmonella enterica subspecies enterica finds its niche in warm-blooded animals and is the primary species associated with human infections. S. bongori and other S. enterica subspecies are more commonly associated with cold-blooded animals, and in some cases can cause disease in these animals (Brenner, et al., 2000).

A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

ABSTRACT Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ70-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10−8 to 10−10 in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCE This article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae. This technology will allow for improved in vivo analysis of bacterial pathogens.

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

ABSTRACT Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia.

Prediction of New Bacterial Type III Secreted Effectors with a Recursive Hidden Markov Model Profile-Alignment Strategy

To identify new bacterial type III secreted effectors is computationally a big challenge. At least a dozen machine learning algorithms have been developed, but so far have only achieved limited success. Sequence similarity appears important for biologists but is frequently neglected by algorithm developers for effector prediction, although large success was achieved in the field with this strategy a decade ago. In this study, we propose a recursive sequence alignment strategy with Hidden Markov Models, to comprehensively find homologs of known YopJ/P full-length proteins, effector domains and N-terminal signal sequences. Using this method, we identified 155 different YopJ/P-family effectors and 59 proteins with YopJ/P N-terminal signal sequences from 27 genera and more than 70 species. Among these genera, we also identified one type III secretion system (T3SS) from Uliginosibacterium and two T3SSs from Rhizobacter for the first time. Higher conservation of effector domains, N-terminal fusion of signal sequences to other effectors, and the exchange of N-terminal signal sequences between different effector proteins were frequently observed for YopJ/P-family proteins. This made it feasible to identify new effectors based on separate similarity screening for the N-terminal signal peptides and the effector domains of known effectors. This method can also be applied to search for homologues of other known T3SS effectors.