Petros Karakousis - Academia.edu (original) (raw)

Papers by Petros Karakousis

Methods: Global gene expression analysis was used to study M. tuberculosis treated with isoniazid... more Methods: Global gene expression analysis was used to study M. tuberculosis treated with isoniazid in dormancy models of nutrient depletion and progressive hypoxia in vitro, as well as in an in vivo hollow fibre model of dormancy. Mycobacterial expression of the drug's putative transcriptional signa- ture was investigated by RT-PCR in each dormancy model, and during the early and chronic

PloS one, 2015

The formation and maintenance of granulomas is central to the host response to Mycobacterium tube... more The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T ...

ACS infectious diseases, Jan 8, 2015

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resist... more Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing...

Tuberculosis, 2015

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our u... more The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p < 0.05) by combining 15 datasets of tuberculosis and other infectious diseases. 15 miRNAs were found to be significantly differentially regulated (7 up-regulated, 8 down-regulated; p < 0.05) by combining 53 datasets of tuberculosis and malignancies. Most of the miRNA signatures identified in this study were found to be involved in immune responses and metabolism. Expression of these miRNA signatures in serum samples from TB subjects (n = 11) as well as healthy controls (n = 10) was examined by TaqMan miRNA array. Taken together, the results revealed differential expression of miRNAs in TB, but available datasets are limited and these miRNA signatures should be validated in future studies.

PloS one, 2011

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition ... more Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosi...

JAMA Ophthalmology, 2014

T uberculosis (TB) remains a global health problem, with almost one-third of the world's populati... more T uberculosis (TB) remains a global health problem, with almost one-third of the world's population being infected. The disease presents primarily as pulmonary TB and, in approximately 20% of patients, as extrapulmonary disease. After Mycobacterium tuberculosis has been inhaled, the organisms undergo phagocytosis by alveolar macrophages, resulting in bacterial containment. 2 In some patients, the bacilli may disseminate systemically, establishing latent infection at extrapulmonary sites, with the potential to reactivate at a later time. 2 Reactivation of latent TB usually occurs when the host's immune system is compromised. 3,4 Extrapulmonary TB is believed to result from reactivation of latent mycobacteria residing within resident reticuloendothelial cells. Ocular TB is a form of extrapulmonary TB manifesting primarily as posterior uveitis. 5 However, the location of latent TB organisms in the eye is not clear. In a study of TB-related posterior uveitis, Rao et al 6 proposed that M tuberculosis may reside in the retinal pigment epithelium (RPE). However, the mechanisms underlying phagocytosis and growth containment of the bacilli by RPE remain unknown. The normal clinical appearance of RPE in individuals before reactivation of M tuberculosis suggests that latent M tuberculosis infection does not induce necrosis or apoptosis.

PLoS ONE, 2012

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tu... more The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allelespecific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

PLOS One, 2010

BackgroundIt has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis ... more BackgroundIt has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.Methodology/Principal FindingsBy testing three M. tuberculosis H37Rv samples, one deletion mutant,

Tuberculosis, 2011

Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS d... more Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS disease. We therefore examined CNS dissemination by different laboratory strains (two M. tuberculosis H37Rv, one CDC1551) in a guinea pig aerosol infection model. Although all strains grew exponentially in lungs, with similar bacterial burdens at the time of extrapulmonary dissemination, M. tuberculosis CDC1551 disseminated to the CNS significantly more than the H37Rv strains. No CNS lesions were observed throughout the study, with only a modest cytokine response. These data suggest that M. tuberculosis may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB.

PLoS ONE, 2010

Background: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosi... more Background: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Mayo Clinic Proceedings, 2001

We report a case of Waterhouse-Friderichsen syndrome associated with group A streptococcus (GAS) ... more We report a case of Waterhouse-Friderichsen syndrome associated with group A streptococcus (GAS) toxic shock syndrome in a previously healthy man. The patient presented with neck pain and fevers of 2 days' duration. Computed tomography of the neck revealed a mass in the retropharyngeal space, suggesting an abscess. Despite prompt treatment with appropriate antibiotics, the patient experienced a fulminant course and died within 8 hours of presentation. Antemortem blood cultures grew GAS positive for exotoxins A, B, and C. Postmortem examination revealed bilateral adrenal hemorrhage, consistent with DIC = disseminated intravascular coagulation; GAS = group A streptococcus Waterhouse-Friderichsen syndrome. Immunohistochemical analysis of the adrenal glands revealed the presence of GAS antigens. However, no disseminated intravascular coagulation was evident. This case demonstrates that adrenal hemorrhage can occur without associated coagulopathy and may result directly from the action of bacterial toxins.

The Lancet Infectious Diseases, 2005

The Lancet Infectious Diseases, 2004

For personal use. Only reproduce with permission from Elsevier Ltd. 557 Disseminated Mycobacteriu... more For personal use. Only reproduce with permission from Elsevier Ltd. 557 Disseminated Mycobacterium avium complex (MAC) infection is a common complication of late-stage HIV-1 infection. Since the advent of highly active antiretroviral therapy (HAART), the rate of MAC infection has declined substantially, but patients with low CD4 cell counts remain at risk. Among patients in the Johns Hopkins cohort with advanced HIV disease, the proportion developing MAC has fallen from 16% before 1996 to 4% after 1996, with a current rate of less than 1% per year. Factors associated with developing MAC include younger age, no use of HAART, and enrolment before 1996.

The Journal of Infectious Diseases, 2009

Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment wit... more Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment within macrophage phagosomes. We studied MTB growth, antibiotic susceptibility, and gene expression during phosphate limitation. With use of MTB mutants deficient in phosphate-related genes, we assessed bacillary survival under phosphate-limited conditions and in mouse and guinea pig lungs. Phosphate limitation restricted MTB growth in a dose-dependent manner, and phosphate-starved bacilli became phenotypically tolerant to isoniazid. The MTB genes ppk1 and relA were upregulated significantly after phosphate starvation, consistent with inorganic polyphosphate accumulation and MTB stringent response induction. The phosphate-specific transport operon pstS3-pstC2-pstA1 was induced during phosphate starvation and its expression was dependent on the 2-component regulatory system SenX3-RegX3. The MTB gene regX3 appears to be essential for bacillary survival during phosphate limitation and in mammalian lungs. Our data suggest that MTB encounters phosphate-limited conditions during mammalian lung infection and that expression of the phosphate starvation response (PSR) is important for MTB persistence.

Journal of Experimental Medicine, 2004

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an import... more Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR ( Rv3133c ) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel Mtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-␥ dependent.

Journal of Bacteriology, 2008

To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculos... more To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N 14-18 -NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N 15 -GGGTTT was identified in the 5 UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

Journal of Bacteriology, 2007

SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent ... more SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions, M. tuberculosis sigF is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in M. tuberculosis entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a sigF-deficient mutant has altered cell membrane properties. Using an M. tuberculosis sigF deletion mutant, we show here that sigF is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress sigF, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of sigF resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following sigF induction was Rv3301c (phoY1), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates phoY1, whose proposed promoter sequence is GGATTG-N 16 -GGGTAT.

Journal of Antimicrobial Chemotherapy, 2007

Objectives: Despite having potent activity against actively replicating Mycobacterium tuberculosi... more Objectives: Despite having potent activity against actively replicating Mycobacterium tuberculosis, isoniazid has very limited activity against dormant bacilli. In order to investigate the lack of bactericidal activity of this drug under conditions leading to mycobacterial dormancy, we studied the transcriptional pattern of M. tuberculosis in different physiological states following exposure to isoniazid.

Methods: Global gene expression analysis was used to study M. tuberculosis treated with isoniazid... more Methods: Global gene expression analysis was used to study M. tuberculosis treated with isoniazid in dormancy models of nutrient depletion and progressive hypoxia in vitro, as well as in an in vivo hollow fibre model of dormancy. Mycobacterial expression of the drug's putative transcriptional signa- ture was investigated by RT-PCR in each dormancy model, and during the early and chronic

PloS one, 2015

The formation and maintenance of granulomas is central to the host response to Mycobacterium tube... more The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T ...

ACS infectious diseases, Jan 8, 2015

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resist... more Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing...

Tuberculosis, 2015

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our u... more The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p < 0.05) by combining 15 datasets of tuberculosis and other infectious diseases. 15 miRNAs were found to be significantly differentially regulated (7 up-regulated, 8 down-regulated; p < 0.05) by combining 53 datasets of tuberculosis and malignancies. Most of the miRNA signatures identified in this study were found to be involved in immune responses and metabolism. Expression of these miRNA signatures in serum samples from TB subjects (n = 11) as well as healthy controls (n = 10) was examined by TaqMan miRNA array. Taken together, the results revealed differential expression of miRNAs in TB, but available datasets are limited and these miRNA signatures should be validated in future studies.

PloS one, 2011

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition ... more Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosi...

JAMA Ophthalmology, 2014

T uberculosis (TB) remains a global health problem, with almost one-third of the world's populati... more T uberculosis (TB) remains a global health problem, with almost one-third of the world's population being infected. The disease presents primarily as pulmonary TB and, in approximately 20% of patients, as extrapulmonary disease. After Mycobacterium tuberculosis has been inhaled, the organisms undergo phagocytosis by alveolar macrophages, resulting in bacterial containment. 2 In some patients, the bacilli may disseminate systemically, establishing latent infection at extrapulmonary sites, with the potential to reactivate at a later time. 2 Reactivation of latent TB usually occurs when the host's immune system is compromised. 3,4 Extrapulmonary TB is believed to result from reactivation of latent mycobacteria residing within resident reticuloendothelial cells. Ocular TB is a form of extrapulmonary TB manifesting primarily as posterior uveitis. 5 However, the location of latent TB organisms in the eye is not clear. In a study of TB-related posterior uveitis, Rao et al 6 proposed that M tuberculosis may reside in the retinal pigment epithelium (RPE). However, the mechanisms underlying phagocytosis and growth containment of the bacilli by RPE remain unknown. The normal clinical appearance of RPE in individuals before reactivation of M tuberculosis suggests that latent M tuberculosis infection does not induce necrosis or apoptosis.

PLoS ONE, 2012

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tu... more The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allelespecific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

PLOS One, 2010

BackgroundIt has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis ... more BackgroundIt has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.Methodology/Principal FindingsBy testing three M. tuberculosis H37Rv samples, one deletion mutant,

Tuberculosis, 2011

Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS d... more Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS disease. We therefore examined CNS dissemination by different laboratory strains (two M. tuberculosis H37Rv, one CDC1551) in a guinea pig aerosol infection model. Although all strains grew exponentially in lungs, with similar bacterial burdens at the time of extrapulmonary dissemination, M. tuberculosis CDC1551 disseminated to the CNS significantly more than the H37Rv strains. No CNS lesions were observed throughout the study, with only a modest cytokine response. These data suggest that M. tuberculosis may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB.

PLoS ONE, 2010

Background: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosi... more Background: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Mayo Clinic Proceedings, 2001

We report a case of Waterhouse-Friderichsen syndrome associated with group A streptococcus (GAS) ... more We report a case of Waterhouse-Friderichsen syndrome associated with group A streptococcus (GAS) toxic shock syndrome in a previously healthy man. The patient presented with neck pain and fevers of 2 days' duration. Computed tomography of the neck revealed a mass in the retropharyngeal space, suggesting an abscess. Despite prompt treatment with appropriate antibiotics, the patient experienced a fulminant course and died within 8 hours of presentation. Antemortem blood cultures grew GAS positive for exotoxins A, B, and C. Postmortem examination revealed bilateral adrenal hemorrhage, consistent with DIC = disseminated intravascular coagulation; GAS = group A streptococcus Waterhouse-Friderichsen syndrome. Immunohistochemical analysis of the adrenal glands revealed the presence of GAS antigens. However, no disseminated intravascular coagulation was evident. This case demonstrates that adrenal hemorrhage can occur without associated coagulopathy and may result directly from the action of bacterial toxins.

The Lancet Infectious Diseases, 2005

The Lancet Infectious Diseases, 2004

For personal use. Only reproduce with permission from Elsevier Ltd. 557 Disseminated Mycobacteriu... more For personal use. Only reproduce with permission from Elsevier Ltd. 557 Disseminated Mycobacterium avium complex (MAC) infection is a common complication of late-stage HIV-1 infection. Since the advent of highly active antiretroviral therapy (HAART), the rate of MAC infection has declined substantially, but patients with low CD4 cell counts remain at risk. Among patients in the Johns Hopkins cohort with advanced HIV disease, the proportion developing MAC has fallen from 16% before 1996 to 4% after 1996, with a current rate of less than 1% per year. Factors associated with developing MAC include younger age, no use of HAART, and enrolment before 1996.

The Journal of Infectious Diseases, 2009

Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment wit... more Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment within macrophage phagosomes. We studied MTB growth, antibiotic susceptibility, and gene expression during phosphate limitation. With use of MTB mutants deficient in phosphate-related genes, we assessed bacillary survival under phosphate-limited conditions and in mouse and guinea pig lungs. Phosphate limitation restricted MTB growth in a dose-dependent manner, and phosphate-starved bacilli became phenotypically tolerant to isoniazid. The MTB genes ppk1 and relA were upregulated significantly after phosphate starvation, consistent with inorganic polyphosphate accumulation and MTB stringent response induction. The phosphate-specific transport operon pstS3-pstC2-pstA1 was induced during phosphate starvation and its expression was dependent on the 2-component regulatory system SenX3-RegX3. The MTB gene regX3 appears to be essential for bacillary survival during phosphate limitation and in mammalian lungs. Our data suggest that MTB encounters phosphate-limited conditions during mammalian lung infection and that expression of the phosphate starvation response (PSR) is important for MTB persistence.

Journal of Experimental Medicine, 2004

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an import... more Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR ( Rv3133c ) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel Mtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-␥ dependent.

Journal of Bacteriology, 2008

To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculos... more To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N 14-18 -NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N 15 -GGGTTT was identified in the 5 UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

Journal of Bacteriology, 2007

SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent ... more SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions, M. tuberculosis sigF is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in M. tuberculosis entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a sigF-deficient mutant has altered cell membrane properties. Using an M. tuberculosis sigF deletion mutant, we show here that sigF is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress sigF, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of sigF resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following sigF induction was Rv3301c (phoY1), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates phoY1, whose proposed promoter sequence is GGATTG-N 16 -GGGTAT.

Journal of Antimicrobial Chemotherapy, 2007

Objectives: Despite having potent activity against actively replicating Mycobacterium tuberculosi... more Objectives: Despite having potent activity against actively replicating Mycobacterium tuberculosis, isoniazid has very limited activity against dormant bacilli. In order to investigate the lack of bactericidal activity of this drug under conditions leading to mycobacterial dormancy, we studied the transcriptional pattern of M. tuberculosis in different physiological states following exposure to isoniazid.