Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

A Streptococcus Quorum Sensing System Enables Suppression of Innate Immunity.

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-ß) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses.IMPORTANCEStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

A Quorum Sensing-Regulated Protein Binds Cell Wall Components and Enhances Lysozyme Resistance in Streptococcus pyogenes.

The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A Streptococcus (GAS; Streptococcus pyogenes). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, spy49_0414c, abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides.IMPORTANCE This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface.

First human implant of the Alterra Adaptive PrestentTM : A new self-expanding device designed to remodel the right ventricular outflow tract.

Current balloon expandable transcatheter valves have limited applicability to patients with "native" right ventricular outflow tracts (RVOT), meaning those who have had previous surgery and are left with large, compliant, irregular RVOT. The Alterra Adaptive PrestentTM is a self-expanding, partially covered stent that was designed to internally reconfigure these types of RVOT, making them suitable for implantation of a commercially available balloon expandable heart valve, the SAPIEN 3. Herein, we describe the first human implant of this device.

PptAB Exports Rgg Quorum-Sensing Peptides in Streptococcus.

A transposon mutagenesis screen designed to identify mutants that were defective in peptide-pheromone signaling of the Rgg2/Rgg3 pathway in Streptococcus pyogenes generated insertions in sixteen loci displaying diminished reporter activity. Fourteen unique transposon insertions were mapped to pptAB, an ABC-type transporter recently described to export sex pheromones of Enterococcus faecalis. Consistent with an idea that PptAB exports signaling peptides, the pheromones known as SHPs (short hydrophobic peptides) were no longer detected in cell-free culture supernatants in a generated deletion mutant of pptAB. PptAB exporters are conserved among the Firmicutes, but their function and substrates remain unclear. Therefore, we tested a pptAB mutant generated in Streptococcus mutans and found that while secretion of heterologously expressed SHP peptides required PptAB, secretion of the S. mutans endogenous pheromone XIP (sigX inducing peptide) was only partially disrupted, indicating that a secondary secretion pathway for XIP exists.

PepO, a CovRS-controlled endopeptidase, disrupts Streptococcus pyogenes quorum sensing.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg(2+) and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles.

Induction of a quorum sensing pathway by environmental signals enhances group A streptococcal resistance to lysozyme.

The human-restricted pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) is responsible for wide-ranging pathologies at numerous sites in the body but has the proclivity to proliferate in individuals asymptomatically. The ability to survive in diverse tissues is undoubtedly benefited by sensory pathways that recognize environmental cues corresponding to stress and nutrient availability and thereby trigger adaptive responses. We investigated the impact that environmental signals contribute to cell-to-cell chemical communication [quorum sensing (QS)] by monitoring activity of the Rgg2/Rgg3 and SHP-pheromone system in GAS. We identified metal limitation and the alternate carbon source mannose as two environmental indicators likely to be encountered by GAS in the host that significantly induced the Rgg-SHP system. Disruption of the metal regulator MtsR partially accounted for the response to metal depletion, whereas ptsABCD was primarily responsible for QS induction due to mannose, but each sensory system induced Rgg-SHP signaling apparently by different mechanisms. Significantly, we found that induction of QS, regardless of the GAS serotype tested, led to enhanced resistance to the antimicrobial agent lysozyme. These results indicate the benefits for GAS to integrate environmental signals with intercellular communication pathways in protection from host defenses.

Satisfaction with care and decision making among parents/caregivers in the pediatric intensive care unit: a comparison between English-speaking whites and Latinos.

PURPOSE: Because of previously documented health care disparities, we hypothesized that English-speaking Latino parents/caregivers would be less satisfied with care and decision making than English-speaking non-Latino white (NLW) parents/caregivers. MATERIALS AND METHODS: An intensive care unit (ICU) family satisfaction survey, Family Satisfaction in the Intensive Care Unit Survey (pediatric, 24 question version), was completed by English-speaking parents/caregivers of children in a cardiothoracic ICU at a university-affiliated children's hospital in 2011. English-speaking NLW and Latino parents/caregivers of patients, younger than 18 years, admitted to the ICU were approached to participate on hospital day 3 or 4 if they were at the bedside for greater than or equal to 2 days. Analysis of variance, χ(2), and Student t tests were used. Cronbach αs were calculated. RESULTS: Fifty parents/caregivers completed the survey in each group. Latino parents/caregivers were younger, more often mothers born outside the United States, more likely to have government insurance or no insurance, and had less education and income. There were no differences between the groups' mean overall satisfaction scores (92.6 ± 8.3 and 93.0 ± 7.1, respectively; P = .80). The Family Satisfaction in the Intensive Care Unit Survey (pediatric, 24 question version) showed high internal consistency reliability (α = .95 and .91 for NLW and Latino groups, respectively). CONCLUSIONS: No disparities in ICU satisfaction with care and decision making between English-speaking NLW and Latino parents/caregivers were found.

The effect of neighborhood and individual characteristics on pediatric critical illness.

The relationship between neighborhood/individual characteristics and pediatric intensive care unit (PICU) outcomes is largely unexplored. We hypothesized that individual-level racial/ethnic minority status and neighborhood-level low socioeconomic status and minority concentration would adversely affect children's severity of illness on admission to the PICU. Retrospective analyses (1/1/2007-5/23/2011) of clinical, geographic, and demographic data were conducted at an academic, tertiary children's hospital PICU. Clinical data included age, diagnosis, insurance, race/ethnicity, Pediatric Index of Mortality 2 score on presentation to the PICU (PIM2), and mortality. Residential addresses were geocoded and linked with 2010 US Census tract data using geographic information systems geocoding techniques. Repeated measures models to predict PIM2 and mortality were constructed using three successive models with theorized covariates including the patient's race/ethnicity, the predominant neighborhood racial/ethnic group, interactions between patient race/ethnicity and neighborhood race/ethnicity, neighborhood socioeconomic status, and insurance type. Of the 5,390 children, 57.8% were Latino and 70.1% possessed government insurance. Latino children (ß = 0.31; p < 0.01), especially Latino children living in a Latino ethnic enclave (ß = 1.13; p < 0.05), had higher PIM2 scores compared with non-Latinos. Children with government insurance (ß = 0.29; p < 0.01) had higher PIM2 scores compared to children with other payment types and median neighborhood income was inversely associated with PIM2 scores (ß = -0.04 per $10,000/year of income; p < 0.05). Lower median neighborhood income, Latino ethnicity, Latino children living in a predominantly Latino neighborhood, and children possessing government insurance were associated with a higher severity of illness on PICU admission. The reasons why these factors affect critical illness severity require further exploration.

Psychometric evaluation of a modified version of the family satisfaction in the ICU survey in parents/caregivers of critically ill children*.

OBJECTIVES: The Family Satisfaction in the Intensive Care Unit 24 (FS-ICU 24) survey consists of two domains (overall care and medical decision-making) and was validated only for family members of adult patients in the ICU. The purpose of this study was to evaluate the internal consistency and construct validity of the FS-ICU 24 survey modified for parents/caregivers of pediatric patients (Pediatric Family Satisfaction in the Intensive Care Unit 24 [pFS-ICU 24]) by comparing it to McPherson's PICU satisfaction survey, in a similar racial/ethnic population as the original Family Satisfaction in the Intensive Care Unit validation studies (English-speaking Caucasian adults). We hypothesized that the pFS-ICU 24 would be psychometrically sound to assess satisfaction of parents/caregivers with critically ill children. DESIGN: A prospective survey examination of the pFS-ICU 24 was performed (1/2011-12/2011). Participants completed the pFS-ICU 24 and McPherson's survey with the order of administration alternated with each consecutive participant to control for order effects (nonrandomized). Cronbach's alphas (α) were calculated to examine internal consistency reliability, and Pearson correlations were calculated to examine construct validity. SETTING: University-affiliated, children's hospital, cardiothoracic ICU. SUBJECTS: English-speaking Caucasian parents/caregivers of children less than 18 years old admitted to the ICU (on hospital day 3 or 4) were approached to participate if they were at the bedside for greater than or equal to 2 days. MEASUREMENTS AND MAIN RESULTS: Fifty parents/caregivers completed the surveys (mean age ± SD = 36.2±9.6 yr; 56% mothers). The α for the pFS-ICU 24 was 0.95 and 0.92 for McPherson's survey. Overall, responses for the pFS-ICU 24 and McPherson's survey were significantly correlated (r = 0.73; p < 0.01). The average overall pFS-ICU 24 satisfaction score was 92.6 ± 8.3. The average pFS-ICU 24 satisfaction with care domain and medical decision-making domain scores were 93.3 ± 8.8 and 91.2 ± 8.9, respectively. CONCLUSIONS: The pFS-ICU 24 is a psychometrically sound measure of satisfaction with care and medical decision-making of parents/caregivers with children in the ICU.

Redundant group a streptococcus signaling peptides exhibit unique activation potentials.

All bacterial quorum sensing (QS) systems are based on the production, secretion, and detection of small signaling molecules. Gram-positive bacteria typically use small peptides as QS effectors, and each QS circuit generally requires the interaction of a single signaling molecule with a single receptor protein. The recently described Rgg2 and Rgg3 (Rgg2/3) regulatory circuit of Streptococcus pyogenes (group A streptococcus [GAS]) is one of only a few QS circuits known to utilize multiple signaling peptides. In this system, two distinct, endogenously produced peptide pheromones (SHP2 and SHP3) both function to activate the QS circuit. The aim of this study was to further define the roles of SHP2 and SHP3 in activation of the Rgg2/3 QS system, specifically with regard to shp gene identity and dosage. Results from our studies using transcriptional reporters and isogenic GAS mutants demonstrate that shp gene dosage does contribute to Rgg2/3 system induction, as decreased gene dosage results in decreased or absent induction. Beyond this, however, data indicate that the shp genes possess distinct potentials for supporting system activation, with shp3 more readily able to support system activation than shp2. Studies using synthetic peptides and shp gene mutants indicate that the disparate activities of endogenous SHPs are due to production, rather than signaling, differences and are conferred by the N-terminal regions rather than the C-terminal signaling regions of the peptides. These data provide evidence that the N-terminal, noneffector sequences of SHP pheromones influence their production efficiencies and thereby the relative activation potentials of endogenous SHPs.

Mood and health-related quality of life among pediatric patients with heart failure.

Adult patients with heart failure (HF) commonly experience depression, with morbid and mortal consequences. However, mood disorders in pediatric patients with HF are poorly understood. This study examined mood and health-related quality of life (HRQOL) in children with HF and compared them cross-sectionally with those of healthy control subjects and heart transplant (Htx) recipients with good heart function. The 62 participants in this study were divided into three groups: HF subjects (n = 15), Htx subjects (n = 23), and healthy control subjects (n = 24). The HF subjects all had chronic HF with a left ventricular ejection fraction lower than 35 %. All the participants completed the Mini-Mental State Examination (MMSE), the Childhood Depression Inventory (CDI), and the Pediatric Quality-of-Life Inventory Cardiac Module (PedsQL CM). Overall, the MMSE scores and CDI subscale scores were similar for all the groups. The HF and Htx participants scored similarly on the PedsQL CM subscales for HRQOL, treatment anxiety, perceived physical appearance, cognitive function, and communication. However, the HF group had a significantly lower HRQOL related to heart problems and treatment than the Htx group. The prevalence of depression among children with HF is not as high as reported in the adult HF literature. However, certain aspects of HRQOL experienced by pediatric HF patients still suffer, especially those related to heart problems and treatment. Health-related QOL tended to be better for the Htx participants than for the HF participants. Exploring developmental and psychosocial outcomes is critical for patients with HF, especially because it has an impact on vital developmental, academic, and social outcomes.

Positive associations between physical and cognitive performance measures in fibromyalgia.

OBJECTIVE: To investigate the associations between perceived physical function (self-report) and physical and cognitive performance (objective assessments) in persons with fibromyalgia (FM). DESIGN: Correlational study. SETTING: Exercise testing laboratory in Southern California. PARTICIPANTS: Community-residing ambulatory adults meeting the American College of Rheumatology 1990 criteria for FM (N=68; mean age, 59.5y). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Composite Physical Function scale, Senior Fitness Test (3 items), Fullerton Advanced Balance scale, 30-foot walk, Trail Making Test parts A and B, Digit Symbol Substitution Test, a composite score of these 3 cognitive measures, attention/executive function composite, processing speed composite, problem solving, inhibition, and episodic memory composite. RESULTS: Hierarchical regression analyses showed that after controlling for age and FM symptoms, better physical performance (based on assessments, not self-report) was associated with higher cognitive function in attention/executive function, processing speed, problem solving, and inhibition. CONCLUSIONS: Researchers should continue to investigate the relationship between physical and cognitive function in both clinical and nonclinical populations, as well as explore changes across time. Because physical activity has been associated with neural improvements, further research may identify whether particular mechanisms, such as neurogenesis, synaptogenesis, or changes in inflammatory marker levels, are involved.

Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development.

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum.

Role of cholesterol in Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as the delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence.

igr Genes and Mycobacterium tuberculosis cholesterol metabolism.

Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M. tuberculosis, is required for cholesterol metabolism. While igr-deficient strains grow identically to the wild type in the presence of short- and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the Delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout infection.

A novel prfA mutation that promotes Listeria monocytogenes cytosol entry but reduces bacterial spread and cytotoxicity.

Listeria monocytogenes is an environmental bacterium that becomes a pathogen following ingestion by a mammalian host. The transition from environmental organism to pathogen requires significant changes in gene expression, including the increased expression of gene products that contribute to bacterial growth within host cells. PrfA is an L. monocytogenes transcriptional regulator that becomes activated upon bacterial entry into mammalian cells and induces the expression of gene products required for virulence. How PrfA activation occurs is not known, however several mutations have been identified that increase PrfA activity in strains grown in vitro (prfA mutations). Here we describe a novel prfA mutation that enhances extracellular PrfA-dependent gene expression but in contrast to prfA mutants inhibits the cytosol-mediated induction of virulence genes. prfA Y154C strains entered cells and escaped from phagosomes with an efficiency similar to wild type bacteria, however the mutation prevented efficient L. monocytogenes actin polymerization and reduced spread of bacteria to adjacent cells. The prfA Y154C mutation severely attenuated bacterial virulence in mice but the mutant strains did generate target antigen specific CD8(+) effector cells. Interestingly, the prfA Y154C mutant was significantly less cytotoxic for host cells than wild type L. monocytogenes. The prfA Y154C mutant strain may therefore represent a novel attenuated strain of L. monocytogenes for antigen delivery with reduced host cell toxicity.

Identification of Mycobacterial genes that alter growth and pathology in macrophages and in mice.

Mycobacterium tuberculosis lives intracellularly, and many facets of its interactions with host cells are not well understood. We screened an M. tuberculosis transposon library for mutants exhibiting reduced ability to kill eukaryotic cells. Four of the mutants identified had insertions in 3 adjacent genes: single insertions in each of 2 acyl-coenzyme A dehydrogenases (fadE) plus 2 unique insertions in a cytochrome P450 homolog. A mutant in which these genes were replaced by allelic exchange was powerfully attenuated in our macrophage viability assay, and there was a striking defect in its ability to grow intracellularly. Interestingly, the difference between wild-type and mutant growth was minimized in activated macrophages. Aerosol infection of mice revealed a lag in growth, delayed dissemination to the spleen, and reduced lung pathology but no difference in persistence. Thus, these genes may be particularly important for events early during infection.

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant.

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.