Virulence Gene Expression In Response Research Articles

Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

Effect of in-water administration of quorum system inhibitors in broilers’ productive performance and intestinal microbiome in a mild necrotic enteritis challenge

ABSTRACT The poultry industry has been facing the impact of necrotic enteritis (NE), a disease caused by the bacterium Clostridium perfringens producing the haemolytic toxin NetB. NE severity may vary from mild clinical to prominent enteric signs causing reduced growth rates and affecting feed conversion ratio. NetB production is controlled by the Agr-like quorum-sensing (QS) system, which coordinates virulence gene expression in response to bacterial cell density. In this study, the peptide-containing cell-free spent media (CFSM) from Enterococcus faecium was tested in NE challenged broilers in two battery cage and one floor pen studies. Results showed a significant reduction of NE mortality. Metagenomic sequencing of the jejunum microbiome revealed no impact of the CFSM on the microbial community, and growth of C. perfringens was unaffected by CFSM in vitro. The expression of QS-controlled virulence genes netB, plc and pfoA was found to be significantly repressed by CFSM during the mid-logarithmic stage of C. perfringens growth and this corresponded with a significant decrease in haemolytic activity. Purified fractions of CFSM containing bioactive peptides were found to cause reduced haemolysis. These results showed that bioactive peptides reduce NE mortality in broilers by interfering with the QS system of C. perfringens and reducing bacterial virulence. Furthermore, the microbiome of C. perfringens-challenged broilers is not affected by quorum sensing inhibitor containing CFSM.

The quorum-sensing peptidic inhibitor rescues host immune system eradication: A novel infectivity mechanism

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

Small RNA coaR contributes to intestinal colonization in Vibrio cholerae via the two-component system EnvZ/OmpR.

Vibrio cholerae is a waterborne bacterium responsible for worldwide outbreaks of acute and fatal cholera. Recently, small regulatory RNAs (sRNAs) have become increasingly recognized as important regulators of virulence gene expression in response to environmental signals. In this study, we determined that two-component system EnvZ/OmpR was required for intestinal colonization in V. cholerae O1 EI Tor strain E12382. Analysis of the characteristics of OmpR revealed a potential binding site in the intergenic region between vc1470 and vc1471, and qRT-PCR showed that expression of the intergenic region increased 5.3-fold in the small intestine compared to LB medium. Race and northern blot assays were performed and demonstrated a new sRNA, coaR (cholerae osmolarity and acidity related regulatory RNA). A ΔcoaR mutant showed a deficient colonization ability in small intestine with CI of 0.15. We identified a target of coaR, tcpI, a negative regulator of the major pilin subunit of TcpA. The ΔtcpI mutant has an increased colonization with CI of 3.16. The expression of coaR increased 2.8-fold and 3.3-fold under relative acidic and hypertonic condition. In summary, coaR was induced under the condition of high osmolarity and acid stress via EnvZ/OmpR and explained that tcpI relieves pH-mediated repression of toxin co-regulated pilus synthesis.

Depletion of Glucose Activates Catabolite Repression during Pneumonic Plague.

Bacterial pathogenesis depends on changes in metabolic and virulence gene expression in response to changes within a pathogen's environment. The plague-causing pathogen, Yersinia pestis, requires expression of the gene encoding the Pla protease for progression of pneumonic plague. The catabolite repressor protein Crp, a global transcriptional regulator, may serve as the activator of pla in response to changes within the lungs as disease progresses. By using gene reporter fusions, the spatial and temporal activation of the crp and pla promoters was measured in a mouse model of pneumonic plague. In the lungs, crp was highly expressed in bacteria found within large aggregates resembling biofilms, while pla expression increased over time independent of the aggregated state. Increased expression of crp and pla correlated with a reduction in lung glucose levels. Deletion of the glucose-specific phosphotransferase system EIIBC (PtsG) of Y. pestis rescued glucose levels in the lungs, resulting in reduced expression of both crp and pla We propose that activation of pla expression during pneumonic plague is driven by an increase of both Crp and cAMP levels following consumption of available glucose in the lungs by Y. pestis Thus, Crp operates as a sensor linking the nutritional environment of the host to regulation of virulence gene expression.IMPORTANCE Using Yersinia pestis as a model for pneumonia, we discovered that glucose is rapidly consumed, leading to a catabolite-repressive environment in the lungs. As a result, expression of the gene encoding the plasminogen activator protease, a target of the catabolite repressor protein required for Y. pestis pathogenesis, is activated. Interestingly, expression of the catabolite repressor protein itself was also increased in the absence of glucose but only in biofilms. The data presented here demonstrate how a bacterial pathogen senses changes within its environment to coordinate metabolism and virulence gene expression.

Temperature-dependent expression of virulence genes in fish-pathogenic bacteria.

Virulence gene expression in pathogenic bacteria is modulated by environmental parameters. A key factor in this expression is temperature. Its effect on virulence gene expression in bacteria infecting warm-blooded hosts is well documented. Transcription of virulence genes in these bacteria is induced upon a shift from low environmental to a higher host temperature (37°C). Interestingly, host temperatures usually correspond to the optimum for growth of these pathogenic bacteria. On the contrary, in ectothermic hosts such as fish, molluscs, and amphibians, infection processes generally occur at a temperature lower than that for the optimal growth of the bacteria. Therefore, regulation of virulence gene expression in response to temperature shift has to be modulated in a different way to that which is found in bacteria infecting warm-blooded hosts. The current understanding of virulence gene expression and its regulation in response to temperature in fish-pathogenic bacteria is limited, but constant extension of our knowledge base is essential to enable a rational approach to the problem of the bacterial fish diseases affecting the aquaculture industry. This is an interesting issue and progress needs to be made in order to diminish the economic losses caused by these diseases. The intention of this review is, for the first time, to compile the scattered results existing in the field in order to lay the groundwork for future research. This article is an overview of those relevant virulence genes that are expressed at temperatures lower than that for optimal bacterial growth in different fish-pathogenic bacteria as well as the principal mechanisms that could be involved in their regulation.

CRH promotes S. pneumoniae growth in vitro and increases lung carriage in mice

Streptococcus pneumoniae (S. pneumoniae), a commensal across the nasal passages, is responsible for the majority of infectious pneumonia cases worldwide. Previous studies have shown that hormonal factors may be influential in regulating S. pneumoniae’s transition from a non-pathogen to a pathogenic state. The current study investigated the effects of corticotropin-releasing hormone (CRH), a peptide hormone involved in stress, on the pathogenicity of S. pneumoniae. Mice were infected with CRH-treated S. pneumoniae via intranasal route, showing an increase in pulmonary bacterial burden. We also quantified S. pneumoniae’s response to CRH through limited serial dilutions and growth curve analysis. We demonstrated that CRH promotes S. pneumoniae titer-dependent proliferation, as well as accelerates log-phase growth. Results also showed an increase in pneumococcal-associated virulence protein A virulence gene expression in response to CRH. These results demonstrate a role for CRH in S. pneumoniae pathogenicity, thus implicating CRH in mediating the transition of S. pneumoniae into a pathogenic state.

Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection

Urinary tract infections (UTIs) are among the most common bacterial infections, causing considerable morbidity in females. Infection is highly recurrent despite appropriate antibiotic treatment. Uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, invades bladder epithelial cells (BECs) and develops into clonal intracellular bacterial communities (IBCs). Upon maturation, IBCs disperse, with bacteria spreading to neighboring BECs to repeat this cycle. This process allows UPEC to gain a foothold in the face of innate defense mechanisms, including micturition, epithelial exfoliation, and the influx of polymorphonuclear leukocytes. Here, we investigated the mechanism and dynamics of urothelial exfoliation in the early acute stages of infection. We show that UPEC α-hemolysin (HlyA) induces Caspase-1/Caspase-4-dependent inflammatory cell death in human urothelial cells, and we demonstrate that the response regulator (CpxR)-sensor kinase (CpxA) two-component system (CpxRA), which regulates virulence gene expression in response to environmental signals, is critical for fine-tuning HlyA cytotoxicity. Deletion of the cpxR transcriptional response regulator derepresses hlyA expression, leading to enhanced Caspase-1/Caspase-4- and NOD-like receptor family, pyrin domain containing 3-dependent inflammatory cell death in human urothelial cells. In vivo, overexpression of HlyA during acute bladder infection induces more rapid and extensive exfoliation and reduced bladder bacterial burdens. Bladder fitness is restored fully by inhibition of Caspase-1 and Caspase-11, the murine homolog of Caspase-4. Thus, we have discovered that fine-tuning of HlyA expression by the CpxRA system is critical for enhancing UPEC fitness in the urinary bladder. These results have significant implications for our understanding of how UPEC establishes persistent colonization.

Vibrio cholerae ToxR Downregulates Virulence Factor Production in Response to Cyclo(Phe-Pro)

ABSTRACTVibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue.

A Temperature-Responsive Network Links Cell Shape and Virulence Traits in a Primary Fungal Pathogen

Author SummaryMicrobial pathogens of humans display the ability to thrive at host temperature. So-called “thermally dimorphic” fungal pathogens, which include Histoplasma capsulatum, are a class of soil fungi that upon being inhaled into the human lung, undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. The ability of these pathogens to cause disease is exquisitely coupled to temperature response. Here we elucidate the regulatory network that governs the ability of H. capsulatum to switch from a filamentous form in the soil environment to a pathogenic yeast form at body temperature. The circuit is driven by three transcription regulators (Ryp1, Ryp2, and Ryp3) that control yeast-phase growth. We show that these factors, which include two highly conserved proteins of the Velvet family of unknown function, bind to specific regulatory DNA elements and directly regulate expression of virulence genes. We identify and characterize Ryp4, a fourth regulator of this pathway, and define DNA motifs that recruit these transcription factors to their temperature-responsive target genes. Our results provide a molecular understanding of how changes in cell shape are linked to expression of virulence genes in thermally dimorphic fungi.

PTS phosphorylation of Mga modulates regulon expression and virulence in the group A streptococcus

The ability of a bacterial pathogen to monitor available carbon sources in host tissues provides a clear fitness advantage. In the group A streptococcus (GAS), the virulence regulator Mga contains homology to phosphotransferase system (PTS) regulatory domains (PRDs) found in sugar operon regulators. Here we show that Mga was phosphorylated in vitro by the PTS components EI/HPr at conserved PRD histidines. A ΔptsI (EI-deficient) GAS mutant exhibited decreased Mga activity. However, PTS-mediated phosphorylation inhibited Mga-dependent transcription of emm in vitro. Using alanine (unphosphorylated) and aspartate (phosphomimetic) mutations of PRD histidines, we establish that a doubly phosphorylated PRD1 phosphomimetic (D/DMga4) is completely inactive in vivo, shutting down expression of the Mga regulon. Although D/DMga4 is still able to bind DNA in vitro, homo-multimerization of Mga is disrupted and the protein is unable to activate transcription. PTS-mediated regulation of Mga activity appears to be important for pathogenesis, as bacteria expressing either non-phosphorylated (A/A) or phosphomimetic (D/D) PRD1 Mga mutants were attenuated in a model of GAS invasive skin disease. Thus, PTS-mediated phosphorylation of Mga may allow the bacteria to modulate virulence gene expression in response to carbohydrate status. Furthermore, PRD-containing virulence regulators (PCVRs) appear to be widespread in Gram-positive pathogens.

Crp Induces Switching of the CsrB and CsrC RNAs in Yersinia pseudotuberculosis and Links Nutritional Status to Virulence

Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome, and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favored when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.

Systematic targeted mutagenesis of the MarR/SlyA family members of Dickeya dadantii 3937 reveals a role for MfbR in the modulation of virulence gene expression in response to acidic pH

Pathogenicity of Dickeya dadantii is a process involving several factors, such as plant cell wall-degrading enzymes and adaptation systems to adverse conditions encountered in the apoplast. Regulators of the MarR family control a variety of biological processes, including adaptation to hostile environments and virulence. Analysis of the members of this family in D. dadantii led to the identification of a new regulator, MfbR, which controls virulence. MfbR represses its own expression but activates genes encoding plant cell wall-degrading enzymes. Purified MfbR increases the binding of RNA polymerase at the virulence gene promoters and inhibits transcription initiation at the mfbR promoter. MfbR activity appeared to be modulated by acidic pH, a stress encountered by pathogens during the early stages of infection. Expression of mfbR and its targets, during infection, showed that MfbR is unable to activate virulence genes in acidic conditions at an early step of infection. In contrast, alkalinization of the apoplast, during an advanced stage of infection, led to the potentialization of MfbR activity resulting in plant cell wall degrading enzyme production. This report presents a new example of how pathogens adjust virulence-associated factors during the time-course of an infection.

Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis

BackgroundWe previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus.ResultsIn this study, we examined the effect of CO2/HCO3-, pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3- increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2- bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested.ConclusionsThis study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

The Mga virulence regulon: infection where the grass is greener

Co-ordinate regulation of virulence gene expression in response to different host environments is central to the success of the group A streptococcus (GAS, Streptococcus pyogenes) as an important human pathogen. Mga represents a ubiquitous stand-alone virulence regulator that controls genes (Mga regulon) whose products are necessary for adherence, internalization and host immune evasion. Mga highly activates a core set of virulence genes, including its own gene, by directly binding to their promoters. Yet, Mga also influences expression of over 10% of the GAS genome, primarily genes and operons involved in metabolism and sugar utilization. Expression of the Mga regulon is influenced by conditions that signify favourable growth conditions, presumably allowing GAS to take advantage of promising new niches in the host. The ability of Mga to respond to growth signals clearly involves regulation of mga expression via global regulatory networks such as RALPs, Rgg/RopB and the catabolite control protein CcpA. However, the presence of predicted PTS regulatory domains (PRDs) within Mga suggests an intriguing model whereby phosphorylation of Mga by the PTS phosphorelay might link growth and sugar utilization with virulence in GAS. As Mga homologues have been found in several important Gram-positive pathogens, the Mga regulon could provide a valuable paradigm for increasing our understanding of global virulence networks in bacteria.

Role for sagA and siaA in Quorum Sensing and Iron Regulation in Streptococcus pyogenes

Streptococcus pyogenes is a ubiquitous and versatile pathogen that causes a variety of infections with a wide range of severity. The versatility of this organism is due in part to its capacity to regulate virulence gene expression in response to the many environments that it encounters during an infection. We analyzed the expression of two potential virulence factors, sagA and siaA (also referred to as pel and htsA, respectively), in response to conditions of varying cell densities and iron concentrations. The sagA gene was up-regulated in conditioned medium from a wild-type strain but not from sagA-deficient mutants, and the gene was also up-regulated in the presence of streptolysin S (SLS), the gene product of sagA, thus indicating that this gene or its product is involved in density-dependent regulation of S. pyogenes. By comparison, siaA responded in a manner consistent with a role in iron acquisition since it was up-regulated under iron-restricted conditions. Although siaA expression was also up-regulated in the presence of SLS and in conditioned media from both wild-type and sagA-deficient mutants, this up-regulation was not growth phase dependent. We conclude that sagA encodes a quorum-sensing signaling molecule, likely SLS, and further support the notion that siaA is likely involved in iron acquisition.

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.

RNA arbitrarily primed PCR survey of genes regulated by ToxR in the deep-sea bacterium Photobacterium profundum strain SS9.

We are currently investigating the role of ToxR-mediated gene regulation in Photobacterium profundum strain SS9. SS9 is a moderately piezophilic ("pressure loving") psychrotolerant marine bacterium belonging to the family Vibrionaceae. In Vibrio cholerae, ToxR is a transmembrane DNA binding protein involved in mediating virulence gene expression in response to various environmental signals. A homolog to V. cholerae ToxR that is necessary for pressure-responsive gene expression of two outer membrane protein-encoding genes was previously found in SS9. To search for additional genes regulated by ToxR in SS9, we have used RNA arbitrarily primed PCR (RAP-PCR) with wild-type and toxR mutant strains of SS9. Seven ToxR-activated transcripts and one ToxR-repressed transcript were identified in this analysis. The cDNAs corresponding to these partial transcripts were cloned and sequenced, and ToxR regulation of their genes was verified. The products of these genes are all predicted to fall into one or both of two functional categories, those whose products alter membrane structure and/or those that are part of a starvation response. The transcript levels of all eight newly identified genes were also characterized as a function of hydrostatic pressure. Various patterns of pressure regulation were observed, indicating that ToxR activation or repression cannot be used to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon in SS9 and indicate that RAP-PCR is a useful approach for the discovery of new genes under the control of global regulatory transcription factors.

Environmental cues and gene expression in Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans.

Microorganisms typically adapt to environmental cues by turning on and off the expression of virulence genes which, in turn, allows for optimal growth and survival within different environmental niches. This adaptation strategy includes sensing and responding to changes in nutrients, pH, temperature, oxygen tension, redox potential, microbial flora, and osmolarity. For a bacterium to adhere to, penetrate, replicate in, and colonize host cells, it is critical that virulence genes are expressed during certain periods of the infection process. Thus, throughout the different stages of an infection, different sets of virulence factors are turned on and off in response to different environmental signals, allowing the bacterium to effectively adapt to its varying niche. In this review, we focus on the regulation of virulence gene expression in two pathogens which have been implicated as major etiological agents in adult and juvenile periodontal diseases: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. Understanding the mechanisms of virulence gene expression in response to the local environment of the host will provide crucial information in the development of effective treatments targeted at eradication of these periodontal disease pathogens.

Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis.

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.