Transcriptional Silencing and Anti-Silencing of Virulence Genes in the Bacterial Pathogen Shigella Flexneri: VIRB, DNA Supercoiling, and the Histone-Like Nucleoid Structuring Protein

Transcriptional silencing and anti-silencing affect many aspects of bacterial physiology, including virulence in bacterial pathogens. In Shigella species, a group of gram-negative pathogens that cause bacillary dysentery in humans, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences virulence genes found on the large virulence plasmid while VirB anti-silences these genes. However, the mechanistic details of their interplay are not fully understood. To elucidate their regulatory mechanisms, I use the icsP virulence locus, which shares a long intergenic region with the divergently transcribed ospZ gene (1535 bp from TSS to TSS). Prior to this work, two discrete H-NS binding regions had been identified, suggesting H-NS-mediated bridging of these two regions as the mechanism of silencing. However, I show that changes to the spacing and helical phasing designed to disrupt the potential bridging were tolerated, suggesting an alternate mechanism of silencing is at play. In addition to H-NS, two other H-NS homologs found in S. flexneri, StpA and Sfh, can also silence the icsP promoter. Interestingly, VirB counters transcriptional silencing mediated by these other H-NS homologs. The site required for VirB-dependent anti-silencing of the icsP promoter is located over 1 kb upstream of the TSS, and nearly 500 bp upstream of the ospZ promoter, but exactly how VirB accomplishes this long-range regulation is not known. I show that VirB docks to this recognition site in vitro and has a high specificity for this site in vivo. Using a combination of 1D and 2D chloroquine-based agarose gel electrophoresis, I demonstrate that, upon docking to its recognition site, VirB triggers a loss of negative supercoiling of our VirB-dependent PicsP-lacZ reporter; importantly, this phenomenon occurs with native VirB levels in S. flexneri. Because H-NS is sensitive to DNA topology at some promoters, it is tantalizing to envision that VirBmediated changes in supercoiling alleviate H-NS-mediated silencing of virulence genes in Shigella. Although anti-silencing proteins in other bacteria, including related pathogens, bear little sequence homology to VirB, the possibility that changes to DNA supercoiling mechanistically unite this group of proteins requires further consideration when studying transcriptional silencing and anti-silencing processes in bacteria

Induction of antibiotic tolerance in bacteria by self-produced and inter-species signaling

Thesis (Ph.D.)--Boston UniversityThough most bacteria within a population are killed by high concentrations of antibiotics, tolerant bacteria survive and can re-grow once antibiotics are removed. Bacterial persisters are dormant cells within an isogenic bacterial population that are tolerant to antibiotic treatment and have been implicated in chronic and recurrent infections. Tolerant and persistent bacteria are generated heterogeneously within populations, and a complete understanding of the processes by which these cells are formed remains elusive. However, there is increasing evidence that bacterial communication by chemical signaling plays a role in establishing population heterogeneity. Here I show that bacterial communication induces persistence in Escherichia coli using the self-produced signaling molecule indole. Indole-induced persister formation was monitored using microfluidics, and oxidative stress and phage-shock pathways were determined to play a role in this phenomenon. I propose a model in which indole signaling "inoculates" a bacterial sub-population against antibiotics by activating stress responses, leading to persister formation. Having demonstrated that communication using the signaling molecule indole controls persistence in the intestinal bacterium E. coli, I sought to determine whether indole could be used as an interspecies signal to control antibiotic tolerance in mixed microbial communities. The common bacterial pathogen Salmonella typhimurium was chosen for these experiments because this species, though closely related to E. coli, does not produce indole. The results demonstrated that indole signaling by E. coli induces tolerance to antibiotics in S. typhimurium. Further, the data suggest that indole-induced tolerance in S. typhimurium is mediated at least in part by the phage shock and oxidative stress response pathways, which were previously implicated in control of indole-induced persistence in E. coli. I used C. elegans as a simple in vivo model for gastrointestinal infection with S. typhimurium, demonstrating that indole signaling increased Salmonella tolerance and altered heterogeneity of infection in this system. These results suggest that antibiotic tolerance in pathogens may be induced by interception of bacterial signals in the host environment

Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens

The innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage. Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat. Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry. The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples. Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity. One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo. We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection. Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia

The Influence of pH on the Survival and Pathogenicity of <i>Salmonella enteritidis</i> Phage-Type 4

An ability to survive the sometimes hostile conditions encountered in food, the environment or a host will be crucial to the pathogenicity of Salmonella enteritidis PT4. Isolates of S. enteritidis and S. typhimurium generally grew under a wider range of stresses when compared to other serotypes of Salmonella; they also survived well in aerosols. S. enteritidis PT4 demonstrated enhanced pH tolerance. Adverse pH was an important discriminatory factor; as this is frequently experienced in nature, the effect of pH on the physiology, tolerance, pathogenicity and proteome expression of a clinical isolate of S. enteritidis PT4 was investigated further. The pH limits for continuous growth (MGT=6.9h), in a minimal medium supplemented with mucin were pH 4.35 and pH 9.45. The adaptive responses of acid and alkaline grown cells differed. Generally, acid grown cells were more tolerant, demonstrating cross-protection to a range of stress factors. In contrast, growth at pH 9.45 induced sensitivity to some stress factors. Growth at the pH limits reduced the percentage of cells expressing fimbriae and/or flagella, and the carriage of the 38MDa plasmid. Cells grown at pH 4.35 were significantly more virulent, both in terms of the number of deaths and the time to death in a mouse model, when compared to pH 7.10 or pH 9.45 grown cells. The proteome of S. enteritidis varied with growth pH when assayed by two-dimensional electrophoresis with N-terminal sequence analysis. The changes were predominantly quantitative with few novel proteins being observed. Proteins which were regulated in response to growth pH included the S. enteritidis homologues to enolase, GroEL and a precursor of the leu/ileu/val binding protein of S. typhlmurium or Escherichia coli. The pH adaptive responses of S. enteritidis PT4 may have a significant role in the life of the cell, by influencing physiology, metabolism, viability and pathogenicity for mice

Functional studies on BolA and related genes: increasing the understanding of a protein with pleiotropic effects

Dissertation presented to obtain a Doctoral degree in Biology by Instituto de Tecnologia Química e BiológicaBolA is a protein that is able to change bacterial shape, confer resistance against large antibiotic molecules and detergents, reduce permeability, change the equilibrium of the outer membrane porins, and it is even involved in biofilm formation. This protein has such pleiotropic effects, that its function has been very difficult to unravel. This was the starting point for the work of this dissertation. If bolA is responsible for global cellular changes that confer resistance to a multitude of stresses, it is imperative to obtain more molecular insights to increase the understanding of the role of BolA in cell physiology and survival.(...)Inês Batista e Guinote was the recipient of a Doctoral Fellowship from Fundação para a Ciência e Tecnologia (FCT): PhD grant – SFRH/BD/ 31758/2006. The work was suspended for 5 months for maternity leave

Genetic screens to identify factors pertinent to host defence against bacterial infection

Antibiotic resistance is a growing concern for healthcare providers across the world. Indeed, resistance is now being found to those ‘last-ditch’ antibiotics reserved for antibiotic-resistant infections. There is great need for research into alternative therapies for globally important pathogens, as well as those that pose a bioterror threat due to their infectivity and high morbidity (e.g. Francisella tularensis). Host-directed therapy as a concept for infection treatment seeks either to block bacterial invasion and growth within host cells or enhance host bactericidal activity. This approach has the potential to treat a broad range of bacterial infections, as well as to reduce the likelihood of developing resistance. As a first step, we explored a host infection response network (including the gene PCDH7) defined on the basis of results from a previous HEK-293 host infection screen. Cellular knockout of human PCDH7 showed reduced intracellular Salmonella enterica serotype Typhimurium (STM) and Shigella sonnei burden in vitro, suggesting resistance to bacterial growth. To identify further targets, a ‘gene trap’ mutation library was generated in a macrophage-like human cell line, U937, which was then differentiated and infected with STM and F. tularensis LVS in independent screens. RNA-Seq was performed on the infected and control populations to identify functionally vital host defence gene mutations. The most statistically significant gene mutations were assessed using pathway analysis tools and literature searches. Multiple pathway analyses converged on electron transport chain subunits MT-ND5, MT-ND6 and MT-CO1, the trapped versions of which were identified as protective in the STM screen. Furthermore, another trapped protective hit, SLC7A11, shows promise from initial validation using a CRISPR knockout (lower intracellular STM burden) as well as pharmacological modulation with the inhibitor sulfasalazine. This work has provided a starting point for the investigation of human genes and cellular processes that might be amenable to pharmacological manipulation to provide protection against, or recovery from, bacterial infection. Therefore host-directed therapies merit further exploration as a novel route to counter the potentially devastating impact of bacteria largely resistant to current antimicrobial drugs.Antibiotic resistance is a growing concern for healthcare providers across the world. Indeed, resistance is now being found to those ‘last-ditch’ antibiotics reserved for antibiotic-resistant infections. There is great need for research into alternative therapies for globally important pathogens, as well as those that pose a bioterror threat due to their infectivity and high morbidity (e.g. Francisella tularensis). Host-directed therapy as a concept for infection treatment seeks either to block bacterial invasion and growth within host cells or enhance host bactericidal activity. This approach has the potential to treat a broad range of bacterial infections, as well as to reduce the likelihood of developing resistance. As a first step, we explored a host infection response network (including the gene PCDH7) defined on the basis of results from a previous HEK-293 host infection screen. Cellular knockout of human PCDH7 showed reduced intracellular Salmonella enterica serotype Typhimurium (STM) and Shigella sonnei burden in vitro, suggesting resistance to bacterial growth. To identify further targets, a ‘gene trap’ mutation library was generated in a macrophage-like human cell line, U937, which was then differentiated and infected with STM and F. tularensis LVS in independent screens. RNA-Seq was performed on the infected and control populations to identify functionally vital host defence gene mutations. The most statistically significant gene mutations were assessed using pathway analysis tools and literature searches. Multiple pathway analyses converged on electron transport chain subunits MT-ND5, MT-ND6 and MT-CO1, the trapped versions of which were identified as protective in the STM screen. Furthermore, another trapped protective hit, SLC7A11, shows promise from initial validation using a CRISPR knockout (lower intracellular STM burden) as well as pharmacological modulation with the inhibitor sulfasalazine. This work has provided a starting point for the investigation of human genes and cellular processes that might be amenable to pharmacological manipulation to provide protection against, or recovery from, bacterial infection. Therefore host-directed therapies merit further exploration as a novel route to counter the potentially devastating impact of bacteria largely resistant to current antimicrobial drugs

The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium

Dissertation presented to obtain the Ph.D degree in BiologyRNAs are important effectors in the process of gene expression. In bacteria, the levels of the transcripts have to be rapidly adjusted in response to constantly changing environmental demands. The cellular concentration of a given RNA is the result of the balance between its synthesis and degradation. RNA degradation is a complex process encompassing multiple pathways. Ribonucleases are the enzymes that directly process and degrade RNA transcripts, regulating their cellular amounts. The rate at which RNA decay occurs depends on the availability of ribonucleases and their specificities according to the sequence and/or the structural elements of the RNA molecule. Several other factors modulate RNA degradation, namely polyadenylation, which plays a multifunctional role in RNA metabolism. Additionally, small non-coding RNAs are crucial regulators of gene expression, and can directly modulate the stability of their mRNA targets. In many cases this regulation is dependent on Hfq, an RNA binding protein which can act in concert with polyadenylation enzymes and is often necessary for the activity of the sRNAs.(...