Location of Pathogenic Bacteria during Persistent Infections: Insights from an Analysis Using Game Theory

Bacterial persistent infections are responsible for a significant amount of the human morbidity and mortality. Unlike acute bacterial infections, it is very difficult to treat persistent bacterial infections (e.g. tuberculosis). Knowledge about the location of pathogenic bacteria during persistent infection will help to treat such conditions by designing novel drugs which can reach such locations. In this study, events of bacterial persistent infections were analyzed using game theory. A game was defined where the pathogen and the host are the two players with a conflict of interest. Criteria for the establishment of Nash equilibrium were calculated for this game. This theoretical model, which is very simple and heuristic, predicts that during persistent infections pathogenic bacteria stay in both intracellular and extracellular compartments of the host. The result of this study implies that a bacterium should be able to survive in both intracellular and extracellular compartments of the host in order to cause persistent infections. This explains why persistent infections are more often caused by intracellular pathogens like Mycobacterium and Salmonella. Moreover, this prediction is in consistence with the results of previous experimental studies.

Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Regulation of respiratory activity of brown adipose tissue mitochondria through changes in cytochromes.

Exposure of cold-acclimatized rats to heat (37 degrees C) for a short period decreased brown adipose tissue (BAT) mitochondrial substrate-dependent oxygen uptake and H2O2 generation. Both the concentration and substrate-dependent rate of cytochrome b reduction decreased as early as 3 h of heat exposure. These results identify cytochrome b as the locus of regulation of electron transport in BAT mitochondria under conditions of heat stress.

Taking Multiple Infections of Cells and Recombination into Account Leads to Small Within-Host Effective-Population-Size Estimates of HIV-1

Whether HIV-1 evolution in infected individuals is dominated by deterministic or stochastic effects remains unclear because current estimates of the effective population size of HIV-1 in vivo, N-e, are widely varying. Models assuming HIV-1 evolution to be neutral estimate N-e similar to 10(2)-10(4), smaller than the inverse mutation rate of HIV-1 (similar to 10(5)), implying the predominance of stochastic forces. In contrast, a model that includes selection estimates N-e>10(5), suggesting that deterministic forces would hold sway. The consequent uncertainty in the nature of HIV-1 evolution compromises our ability to describe disease progression and outcomes of therapy. We perform detailed bit-string simulations of viral evolution that consider large genome lengths and incorporate the key evolutionary processes underlying the genomic diversification of HIV-1 in infected individuals, namely, mutation, multiple infections of cells, recombination, selection, and epistatic interactions between multiple loci. Our simulations describe quantitatively the evolution of HIV-1 diversity and divergence in patients. From comparisons of our simulations with patient data, we estimate N-e similar to 10(3)-10(4), implying predominantly stochastic evolution. Interestingly, we find that N-e and the viral generation time are correlated with the disease progression time, presenting a route to a priori prediction of disease progression in patients. Further, we show that the previous estimate of N-e>10(5) reduces as the frequencies of multiple infections of cells and recombination assumed increase. Our simulations with N-e similar to 10(3)-10(4) may be employed to estimate markers of disease progression and outcomes of therapy that depend on the evolution of viral diversity and divergence.

Nonlinear measures of respiration: Respiratory irregularity and increased chaos of respiration in patients with panic disorder

Background. Respiratory irregularity has been previously reported in patients with panic disorder using time domain measures. However, the respiratory signal is not entirely linear and a few previous studies used approximate entropy (APEN), a measure of regularity of time series. We have been studying APEN and other nonlinear measures including a measure of chaos, the largest Lyapunov exponent (LLE) of heart rate time series, in some detail. In this study, we used these measures of respiration to compare normal controls (n = 18) and patients with panic disorder (n = 22) in addition to the traditional time domain measures of respiratory rate and tidal volume. Methods: Respiratory signal was obtained by the Respitrace system using a thoracic and an abdominal belt, which was digitized at 500 Hz. Later, the time series were constructed at 4 Hz, as the highest frequency in this signal is limited to 0.5 Hz. We used 256 s of data (1,024 points) during supine and standing postures under normal breathing and controlled breathing at 12 breaths/min. Results: APEN was significantly higher in patients in standing posture during normal as well as controlled breathing (p = 0.002 and 0.02, respectively). LLE was also significantly higher in standing posture during normal breathing (p = 0.009). Similarly, the time domain measures of standard deviations and the coefficient of variation (COV) of tidal volume (TV) were significantly higher in the patient group (p = 0.02 and 0.004, respectively). The frequency of sighs was also higher in the patient group in standing posture (p = 0.02). In standing posture, LLE (p < 0.05) as well as APEN (p < 0.01) contributed significantly toward the separation of the two groups over and beyond the linear measure, i.e. the COV of TV. Conclusion: These findings support the previously described respiratory irregularity in patients with panic disorder and also illustrate the utility of nonlinear measures such as APEN and LLE as additional measures toward a better understanding of the abnormalities of respiratory physiology in similar patient populations as the correlation between LLE, APEN and some of the time domain measures only explained up to 50-60% of the variation. Copyright (C) 2002 S. Karger AG, Basel.

Modelling and classification of respiratory volume wavefourms

A technique is proposed for classifying respiratory volume waveforms(RVW) into normal and abnormal categories of respiratory pathways. The proposed method transforms the temporal sequence into frequency domain by using an orthogonal transform, namely discrete cosine transform (DCT) and the transformed signal is pole-zero modelled. A Bayes classifier using model pole angles as the feature vector performed satisfactorily when a limited number of RVWs recorded under deep and rapid (DR) manoeuvre are classified.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

IgG2 subclass isotype antibody and intrauterine infections

The foetus is dependent on its mother for passive immunity involving receptor-mediated specific transport of antibodies. IgG antibody is present in highest concentration in serum and is the only antibody type that can cross the placenta efficiently, except for its IgG2 subclass. Most of the pathogenic manifestations affecting the foetus involve capsular antigens and polysaccharides of pathogens and it is known that immune response to these antigens is primed to the predominant production of IgG2 type of antibody. Paradoxically, the IgG2 subclass cannot cross the placenta and neutralize such antigens; therefore, infections related to these antigens may persist and can lead to serious conditions like miscarriage and stillbirth. This article describes in brief the properties of IgG subclasses, intrauterine infections seen during pregnancy and discusses possible IgG-based strategies to manage infections to afford protection to the foetus.

Lipid coated mesoporous silica nanoparticles as an oral delivery system for targeting and treatment of intravacuolar Salmonella infections

Lipid coated mesoporous silica nanoparticle (L-MSN) were synthesized for oral delivery of ciprofloxacin for intracellular elimination of Salmonella pathogen. The particle size was found to be between 50-100 nm with a lipid coat of approximately 5 nm thickness. The lipid coating was achieved by sonication of liposomes with the MSN particles and evaluated by CLSMand FTIR studies. The L-MSN particles exhibited lower cytotoxicity compared to bare MSN particles. Ciprofloxacin, a fluoroquinolone antibiotic, loaded into the L-MSN particles showed enhanced antibacterial activity against free drug in in vitro assays. The lipid coat was found to aid in intravacuolar targeting of the drug cargo as observed by confocal microscopy studies. We also observed that a lower dose of antibiotic was sufficient to clear the pathogen from mice and increase their survivability using the L-MSN oral delivery system.

Successful treatment of biofilm infections using shock waves combined with antibiotic therapy

Many bacteria secrete a highly hydrated framework of extracellular polymer matrix on suitable substrates and embed within the matrix to form a biofilm. Bacterial biofilms are observed on many medical devices, endocarditis, periodontitis and lung infections in cystic fibrosis patients. Bacteria in biofilm are protected from antibiotics and >1,000 times of the minimum inhibitory concentration may be required to treat biofilm infections. Here, we demonstrated that shock waves could be used to remove Salmonella, Pseudomonas and Staphylococcus biofilms in urinary catheters. The studies were extended to a Pseudomonas chronic pneumonia lung infection and Staphylococcus skin suture infection model in mice. The biofilm infections in mice, treated with shock waves became susceptible to antibiotics, unlike untreated biofilms. Mice exposed to shock waves responded to ciprofloxacin treatment, while ciprofloxacin alone was ineffective in treating the infection. These results demonstrate for the first time that, shock waves, combined with antibiotic treatment can be used to treat biofilm infection on medical devices as well as in situ infections.

Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual-and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.