Petros Karakousis

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveo...

ABSTRACTStatins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. We investigated the mechanism of anti-tubercular action of simvastatin in Mycobacterium tuberculosis-infected human monocytic cells. We found that the anti-tubercular activity of statins was phenocopied by cholesterol-branch but not prenylation-branch inhibitors. Moreover, statin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Overall, our data demonstrate that the anti-tubercular activity of simvastatin r...

Nonadrenergic, noncholinergic (NANC) relaxations of airway smooth muscle are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Previous studies of the parasympathetic innervation of guinea pig trachealis suggest that the ganglion neurons mediating NANC relaxations but not cholinergic contractions are associated with the esophagus. In this study, the location of the neurons mediating these responses and their neurochemical phenotype was further assessed. Guinea pig tracheas maintained in organotypic culture for 2 days with the adjacent esophagus intact displayed cholinergic contractions and NANC relaxations to electrical field stimulation (EFS) as well as VIP and NO synthase (NOS) nerve fiber densities that were similar to those of control tracheas. By contrast, in tracheas cultured without the esophagus, NANC relaxations to EFS were not observed, and VIP and NOS nerve fiber densities were reduced > 80%. EFS-induced cholinergic contractions were ...

is the etiologic agent of tuberculosis. The demand for new chemotherapeutics with unique mechanisms of action to treat (multi)resistant strains is an urgent need. The objective of this work was to test the effect of manganese(II) and copper(II) phenanthroline/dicarboxylate complexes against . The water-soluble Mn(II) complexes, [Mn(oda)(phen)(HO)][Mn(oda)(phen)(oda)]·4HO () and {[Mn(3,6,9-tdda)(phen)]·3HO·EtOH}n () (odaH = octanedioic acid, phen = 1,10-phenanthroline, tddaH = 3,6,9-trioxaundecanedioic acid), and water-insoluble complexes, [Mn(ph)(phen)(HO)] (), [Mn(ph)(phen)(HO)]·4HO (), [Mn(isoph)(phen)]·4HO (), {[Mn(phen)(HO)]}(isoph)(phen)·12HO () and [Mn(tereph)(phen)]·5HO () (phH = phthalic acid, isophH = isophthalic acid, terephH = terephthalic acid), robustly inhibited the viability of strains, H37Rv and CDC1551. The water-soluble Cu(II) analog of (), [Cu(oda)(phen)](ClO)·2.76HO·EtOH (), was significantly less effective against both strains. Whilst () retarded H37Rv growth mu...

Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PA...

There is controversy regarding the potential fitness costs of rifampicin (RIF) resistance-conferring mutations in the Mycobacterium tuberculosis (Mtb) rpoB gene. We characterized the pathogenicity of an Mtb RpoB H526D mutant. A mutant containing the RpoB H526D mutation was isolated from wild-type Mtb grown on RIF-containing plates and complemented for determination of in vitro and in vivo fitness costs. The RpoB H526D mutant showed reduced survival relative to control strains during progressive hypoxia and delayed growth following resuscitation from nutrient starvation (p < 0.05), which was associated with reduced expression of the resuscitation-promoting factor genes rpfB, rpfC and rpfE. Relative to the isogenic wild-type strain, the mutant showed significantly attenuated growth and long-term survival as well as reduced inflammation in mouse lungs. Conclusion & future perspective: Our data suggest that RpoB H526D mutation confers a fitness cost during growth-limiting conditions ...

Candida species are the most common cause of invasive fungal infections, accounting for 70% to 90% of hospital cases.1,2 Although the overall US incidence of candidemia is low among nonneutropenic, critically ill adults (approximately 2/1000 intensive care unit [ICU] admissions), candidemia results in a longer length of stay (≤1 month), costs in excess of $40 000 per case, and a 30-day mortality greater than 50%.1-3 Moreover, mortality from invasive fungal infection has been increasing over the past decade, suggesting that advances in the management for invasive fungal infection have lagged behind those for bacterial septic shock.4 In light of the high mortality associated with invasive fungal infection, particularly among critically ill patients, a number of guidelines have focused on empirical treatment with antifungal echinocandins and surveillance for Candida either through culture or diagnostic biomarkers.5,6 Accordingly, the use of echinocandins has increased from 4.6% to 48.5% in some settings.7 Yet little was previously known about mortality benefits from prophylactic antifungal therapy. A series of recent studies have examined empirical antifungal therapy among nonneutropenic patients with septic shock but have had mixed results concerning invasive fungal infection. In a placebo-controlled trial of empirical fluconazole treatment among critically ill surgical patients, Pelz et al demonstrated a 55% decrease in the rate of invasive fungal infection.8 Schuster et al conducted a similar study using fluconazole among critically ill ICU patients at high risk for invasive candidiasis, but did not find a decrease in the rate of invasive fungal infection.9 Ostrosky-Zeichner et al found a nonsignificant reduction in invasive fungal infection using capsofungin as antifungal prophylaxis among ICU patients with at least 2 risk factors for candidemia.10 However, none of these trials took into account Candida colonization as an inclusion criterion. In this issue of JAMA, Timsit and colleagues11 report findings of a multicenter, randomized, placebo-controlled trial (EMPIRICUS) to determine if a 14-day empirical course of micafungin led to a higher 28-day survival without proven invasive fungal infection relative to placebo in 260 nonneutropenic, nontransplanted, critically ill adult patients with at least 1 colonization site positive for Candida species. In this trial, the authors did not find a difference in the proportion of participants who were alive and without invasive fungal infection at 28 days when compared between the micafungin (68%) and placebo groups (60.2%). Moreover, even though there was a decrease in incident invasive fungal infection among patients who received micafungin (12%) when compared with placebo (3%), there was no difference in associated mortality at 28 days (30% mortality for the micafungin group vs 29.7% for placebo). Although the rates of invasive fungal infection may decrease with targeted administration of empirical therapy, the results of the EMPIRICUS trial together with previous randomized trials demonstrate substantial evidence that empirical antifungal therapy does not confer a mortality benefit. These findings highlight 2 emerging themes in critical care medicine, which are that less is more and the importance of targeted therapies in treating invasive fungal infection. The EMPIRICUS trial was conducted in 19 ICUs in France. The study was well powered to assess difference in mortality between groups and the results are largely generalizable to critically ill patients with characteristics similar to the study inclusion criteria. Nonsignificant improvement in survival was noted among patients with high Sequential Organ Failure Assessment scores, which may suggest that certain critically ill subgroups may benefit from empirical therapy. Additionally, there was low participation among patients with postoperative gastrointestinal leakage and acute necrotizing pancreatitis, among which there is a high risk of invasive fungal infection. Previous studies have shown surgical patients may benefit from empirical therapy.8 Following the development of dedicated critical care units in the 1950s, critical care medicine was characterized as aggressive with maximum interventions for life-saving therapy. If some care is good, then more care was thought to be better.12 The near ubiquitous use of pulmonary artery catheters for invasive monitoring exemplified this approach until a number of studies demonstrated lack of clinical benefit to continuous monitoring and potential iatrogenic risk.12 Over the past 20 years, aggressive goals for many of the most relevant treatment options for critical illness have been revisited and modified or abandoned. Key examples include changes in the following treatment options: (1) the shift from high to low tidal volume for respiratory support; (2) from liberal to conservative strategies for fluid management and blood transfusion therapy; (3) from routine to…

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 ...

W egener's granulomatosis (WG) is a multisystem disease of unknown pathogenesis characterized by necrotizing granulomas of the upper and lower respiratory tract, systemic small-vessel vasculitis and focal necrotizing glomerulonephritis.1 Upper or lower respiratory tract involvement develops in all patients, and 85% of patients eventually manifest renal involvement, which heralds a worse prognosis.2 Ophthalmic disease is the presenting feature in nearly one-sixth of patients and ultimately develops in most cases. 1 Straatsma3 classified the ocular manifestations in WG as contiguous or focal. Contiguous ocular involvement results from direct spread of paranasal sinus disease and may lead to nasolacrimal duct obstruction, proptosis, and orbital abscess or cellulitis. Focal ocular disease may present as conjunctivitis, episcleritis, scleritis, comeoscleral ulceration, uveitis and granulomatous vasculitis of the retina and optic nerve.4 The most common ophthalmic presentation is proptosis from orbital inflammation. 36 Prompt recognition of ocular involvement is critical as approximately 8% of patients with WG permanently lose vision, 1 but the clinical distinction from other entities in the early phase of the disease is often difficult.

The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including "latency," "persistence," "dormancy," and "antibiotic tolerance." Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, "dormant" bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4(+) and CD8(+) T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance ...

Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with...

Mycobacterium tuberculosis (Mtb) must adapt to various stress conditions during host infection. The two-component regulatory system (2CRS) SenX3-RegX3 is required for Mtb virulence. We showed recently that the senX3-regX3 intergenic region contains promoter activity, driving senX3-independent regX3 expression. In the current study, we tested the hypothesis that RegX3 has a SenX3-independent role in Mtb virulence. The gene expression patterns, growth, and survival of mutants containing transposon insertions in senX3 (senX3::Tn) and regX3 (regX3::Tn) were compared to those of their respective complemented strains and the isogenic wild-type parent strain during axenic growth in nutrient-rich broth, phosphate depletion, nutrient starvation, and in the lungs of BALB/c mice. regX3 expression was reduced in senX3::Tn during phosphate depletion and nutrient starvation, and expression of the phosphate-specific transport gene pstC2 was reduced similarly in senX3::Tn and regX3::Tn during phosp...

The bicyclic nitroimidazole-like molecule PA-824 has activity both against replicating and hypoxic non-replicating Mycobacterium tuberculosis, raising the possibility that it may have a role in the treatment of latent tuberculosis infection (LTBI). This study aimed to examine the bactericidal and sterilising activities of PA-824 against LTBI in C3HeB/FeJ mice, which develop hypoxic, necrotic granulomas histologically resembling their human counterparts. Female 5-6-week-old C3HeB/FeJ mice were immunised via the aerosol route with a recombinant BCG strain overexpressing the 30-kDa major secretory protein (rBCG30) and were aerosol-infected 6 weeks later with virulent M. tuberculosis H37Rv. Six weeks after M. tuberculosis infection, separate groups of mice were left untreated (negative controls) or were treated with either rifampicin, isoniazid (INH) or PA-824. Culture-positive relapse was assessed in subgroups of mice after 2 months and 4 months of treatment. Human-equivalent doses of ...

We recently reported that in lung tissue, thioridazine accumulates at high concentrations relative to serum levels, displaying modest synergy with isoniazid and reducing the emergence of isoniazid-resistant mutants in mouse lungs. In this study, we sought to investigate the sterilizing activity of human-equivalent doses of thioridazine when given in combination with the "Denver regimen" against acute murine tuberculosis. We found a trend toward a positive impact of thioridazine on the bacterial clearance and lowering relapse rates of the combined standard TB chemotherapy.

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal mo...

The repurposing of existing drugs is being pursued as a means by which to accelerate the development of novel regimens for the treatment of drug-susceptible and drug-resistant tuberculosis (TB). In the current study, we assessed the activity of the antipsychotic drug thioridazine (TRZ) in combination with the standard regimen in a well-validated murine TB model. Single-dose and steady-state pharmacokinetic studies were performed in BALB/c mice to establish human-equivalent doses of TRZ. To determine the bactericidal activity of TRZ against TB in BALB/c mice, three separate studies were performed, including a dose-ranging study of TRZ monotherapy and efficacy studies of human-equivalent doses of TRZ with and without isoniazid (INH) or rifampin (RIF). Therapeutic efficacy was assessed by the change in mycobacterial load in the lung. The human-equivalent dose of thioridazine was determined to be 25 mg/kg of body weight, which was well tolerated in mice. TRZ was found to accumulate at h...

The need to develop new, improved treatments for tuberculosis (TB) remains urgent, and the repurposing of existing drugs represents a possible shortcut to market. Recently, there has been significant interest in host-directed adjuvant therapy to enhance bacillary killing. HMG-CoA reductase inhibitors (statins), which are among the most commonly prescribed drugs, have immunomodulatory properties and improve the clinical outcomes of bacterial infections. We studied the tuberculocidal activity of simvastatin alone and in combination with first-line anti-TB drugs in J774 macrophages and during chronic TB infection. Exposure to 5 μM simvastatin significantly increased the tuberculocidal activity of isoniazid in J774 macrophages at Day 3 after infection versus isoniazid alone (P=0.02). Similarly, relative to the standard oral regimen of rifampicin (10 mg/kg), isoniazid (10 mg/kg) and pyrazinamide (150 mg/kg) given five times weekly, the addition of 25 mg/kg simvastatin enhanced bacillary ...

Tuberculosis (TB) is a communicable disease of major global importance and causes metabolic disorder of the patients. In a previous study, we found that the plasma metabolite profile of TB patients differs from that of healthy control subjects based on nuclear magnetic resonance (NMR) spectroscopy. In order to evaluate the TB specificity of the metabolite profile, a total of 110 patients, including 40 with diabetes, 40 with malignancy, and 30 with community-acquired pneumonia (CAP), assessed by NMR spectroscopy, and compared to those of patients with TB. Based on the orthogonal partial least-squares discriminant analysis (OPLS-DA), the metabolic profiles of these diseases were significant different, as compared to the healthy controls and TB patients, respectively. The score plots of the OPLS-DA model demonstrated that TB was easily distinguishable from diabetes, CAP and malignancy. Plasma levels of ketone bodies, lactate, and pyruvate were increased in TB patient compared to health...

ABSTRACT Background: Alternative RNA polymerase sigma factors coordinate gene regulation in many bacteria in response to stress conditions. M. tuberculosis (M. tb) sigF is strongly induced during stationary growth, and upon exposure to nutrient starvation and several anti-TB drugs. Also, sigF-deficient M. tb exhibits reduced immunopathology and lethality in mice. In order to gain insight into molecular pathways regulated by sigF, we studied the transcriptional profile of M. tb following sigF overexpression. Methods: An acetamidase promoter-sigF fusion gene and kanamycin resistance gene were integrated into M. tb CDC1551 using plasmid pSCW35 by site-specific recombination (sigF-inducible strain). A control strain of was generated by the same method using plasmid pSCW38 (identical to pSCW38 but lacking the sigF gene). Log-phase cultures of each strain were exposed to 0.2% acetamide for the following time intervals prior to RNA extraction: 0, 6, 12, and 24 h. Microarray analysis was performed using 70-mer oligonucleotides representing all annotated CDC1551 genes. Quantitative RT-PCR was performed for a subset of genes found to be significantly regulated by microarrays. Each strain was exposed to a range of concentrations of isoniazid and rifampin following acetamide induction in vitro to determine drug minimum inhibitory concentrations. Results: Overexpression of sigF did not alter M. tb growth kinetics over a 7 day period in vitro, or susceptibility to the first-line anti-TB drugs isoniazid and rifampin. Microarray analysis and quantitative RT-PCR showed significant upregulation of several genes encoding transcriptional regulatory proteins, including phoY1 and Rv0328, the anti-SigF gene usfX, and several genes encoding conserved hypothetical proteins. The putative SigF consensus recognition sequence GTTTX17GGGTAT was identified upstream of each of these genes. Conclusion: SigF directly and indirectly regulates several important M. tb genes which might play a role in the adaptation of the organism to a variety of stress conditions.

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...

This paper reports a case of nosocomial pneumococcal cellulitis that developed following a lymph-node biopsy in a woman being treated with high-dose intravenous corticosteroids for systemic lupus erythematosis (SLE). Her rapid and severe clinical deterioration was similar to that caused by group A streptococcus. The risk factors for the development of nosocomial pneumococcal cellulitis as a complication of SLE are reviewed and preventive measures discussed.

The highly successful pathogen Mycobacterium tuberculosis (Mtb) has evolved strategies to adapt to various stress conditions, thus promoting survival within the infected host. The two-component regulatory system (2CRS) senX3-regX3, which has been implicated in the Mtb response to inorganic phosphate depletion, is believed to behave as an auto-regulatory bicistronic operon. Unlike other 2CRS, Mtb senX3-regX3 features an intergenic region (IR) containing several mycobacterium interspersed repetitive units (MIRU) of unknown function. In this study, we used a lacZ reporter system to study the promoter activity of the 5′ untranslated region of senX3, and that of various numbers of MIRUs in the senX3-regX3 IR, during axenic Mtb growth in nutrient-rich broth, and upon exposure to growth-restricting conditions. Activity of the senX3 promoter was induced during phosphate depletion and nutrient starvation, and IR promoter activity under these conditions was directly proportional to the number...

Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo . Finally, the new results implicated additional high-priority candidate genes for testi...

The Mycobacterium tuberculosis gene Rv3232c / MT3329 ( ppk2 ) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2 -deficient mutant ( ppk2 ::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2 ::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2 ::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT...