Erin R. Murphy

BhuR, a Virulence-Associated Outer Membrane Protein of Bordetella avium, Is Required for the Acquisition of Iron from Heme and Hemoproteins

ABSTRACT Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

ABSTRACT Efficient utilization of heme as an iron (Fe) source byBordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized thatrhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuIwas correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal tobhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced intoEscherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. colidemonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of therhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5′ untranslated region. In addition, we have identified similar nucleotide sequences within the 5′ untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5′ untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source.

Virf-independent regulation of shigella virB transcription is mediated by the small RNA RyhB

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression.

The Iron-Responsive Fur/RyhB Regulatory Cascade Modulates the Shigella Outer Membrane Protease IcsP

ABSTRACT Actin-based motility is central to the pathogenicity of the intracellular bacterial pathogen Shigella. Two Shigella outer membrane proteins, IcsA and IcsP, are required for efficient actin-based motility in the host cell cytoplasm, and the genes encoding both proteins are carried on the large virulence plasmid. IcsA triggers actin polymerization on the surface of the bacterium, leading to the formation of an actin tail that allows both intra- and intercellular spread. IcsP, an outer membrane protease, modulates the amount and distribution of the IcsA protein on the bacterial surface through proteolytic cleavage of IcsA. Transcription of icsP is increased in the presence of VirB, a DNA-binding protein that positively regulates many genes carried on the large virulence plasmid. In Shigella dysenteriae, the small regulatory RNA RyhB, which is a member of the iron-responsive Fur regulon, suppresses several virulence-associated phenotypes by downregulating levels of virB in response to iron limitation. Here we show that the Fur/RyhB regulatory pathway downregulates IcsP levels in response to low iron concentrations in Shigella flexneri and that this occurs at the level of transcription through the RyhB-dependent regulation of VirB. These observations demonstrate that in Shigella species the Fur/RyhB regulatory pathway provides a mechanism to finely tune the expression of icsP in response to the low concentrations of free iron predicted to be encountered within colonic epithelial cells.

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

Shigella Iron Acquisition Systems and their Regulation

Survival of Shigella within the host is strictly dependent on the ability of the pathogen to acquire essential nutrients, such as iron. As an innate immune defense against invading pathogens, the level of bio-available iron within the human host is maintained at exceeding low levels, by sequestration of the element within heme and other host iron-binding compounds. In response to sequestration mediated iron limitation, Shigella produce multiple iron-uptake systems that each function to facilitate the utilization of a specific host-associated source of nutrient iron. As a mechanism to balance the essential need for iron and the toxicity of the element when in excess, the production of bacterial iron acquisition systems is tightly regulated by a variety of molecular mechanisms. This review summarizes the current state of knowledge on the iron-uptake systems produced by Shigella species, their distribution within the genus, and the molecular mechanisms that regulate their production.

Riboregulators: Fine-Tuning Virulence in Shigella

Within the past several years, RNA-mediated regulation (ribo-regulation) has become increasingly recognized for its importance in controlling critical bacterial processes. Regulatory RNA molecules, or riboregulators, are perpetually responsive to changes within the micro-environment of a bacterium. Notably, several characterized riboregulators control virulence in pathogenic bacteria, as is the case for each riboregulator characterized to date in Shigella. The timing of virulence gene expression and the ability of the pathogen to adapt to rapidly changing environmental conditions is critical to the establishment and progression of infection by Shigella species; ribo-regulators mediate each of these important processes. This mini review will present the current state of knowledge regarding RNA-mediated regulation in Shigella by detailing the characterization and function of each identified riboregulator in these pathogens.

Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis

Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule, RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the overproduction of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread as well as the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1, the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

Transcriptional and posttranscriptional regulation of Shigella shuT in response to host-associated iron availability and temperature

Like most bacteria, Shigella must maintain a precise balance between the necessity and toxicity of iron; a balance that is achieved, at least in part, by regulating the production of bacterial iron acquisition systems in response to specific environmental signals. Using the Shigella heme utilization (Shu) system, S. dysenteriae is able to acquire iron from heme, a potentially rich source of nutritional iron within the otherwise iron‐limited environment of the human host. Investigations presented within reveal two distinct molecular mechanisms underlying previously uncharacterized transcriptional and translational regulation of shuT, a gene encoding the periplasmic‐binding component of the Shu system. While shuT transcription is regulated in response to iron availability via a process dependent upon the global regulator Fur and a Fur‐binding site located immediately downstream of the promoter, shuT translation is regulated in response to environmental temperature via the activity of an RNA thermometer located within the 5′ untranslated region of the gene. Such complex regulation likely increases the fitness of S. dysenteriae by ensuring maximal ShuT production when the pathogen is within the iron‐limited and relatively warm environment of the infected host, the only environment in which heme will be encountered as a potential source of essential iron.