Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.

Prokaryotic toxin-antitoxin stress response loci.

SUMMARY An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Replication and control of circular bacterial plasmids.

Bacteria and fungi can form a range of physical associations that depend on various modes of molecular communication for their development and functioning. These bacterial-fungal interactions often result in changes to the pathogenicity or the nutritional influence of one or both partners toward plants or animals (including humans). They can also result in unique contributions to biogeochemical cycles and biotechnological processes. Thus, the interactions between bacteria and fungi are of central importance to numerous biological questions in agriculture, forestry, environmental science, food production, and medicine. Here we present a structured review of bacterial-fungal interactions, illustrated by examples sourced from many diverse scientific fields. We consider the general and specific properties of these interactions, providing a global perspective across this emerging multidisciplinary research area. We show that in many cases, parallels can be drawn between different scenarios in which bacterial-fungal interactions are important. Finally, we discuss how new avenues of investigation may enhance our ability to combat, manipulate, or exploit bacterial-fungal complexes for the economic and practical benefit of humanity as well as reshape our current understanding of bacterial and fungal ecology.

Bacterial-Fungal Interactions: Hyphens between Agricultural, Clinical, Environmental, and Food Microbiologists

Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

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Efflux-Mediated Antifungal Drug Resistance

The human body plays host to interactions between a diverse range of microorganisms, including bacteria and fungi. In this Review, Mylonakis and colleagues describe the characteristics of medically important bacterial–fungal interactions and highlight how imbalances in these interactions can contribute to human disease. Whether it is in the setting of disease or in a healthy state, the human body contains a diverse range of microorganisms, including bacteria and fungi. The interactions between these taxonomically diverse microorganisms are highly dynamic and dependent on a multitude of microorganism and host factors. Human disease can develop from an imbalance between commensal bacteria and fungi or from invasion of particular host niches by opportunistic bacterial and fungal pathogens. This Review describes the clinical and molecular characteristics of bacterial–fungal interactions that are relevant to human disease.

Medically important bacterial–fungal interactions

http://tmp.wustl.edu/archives/TMPSpring2006/5-24-06SSJBCMukhop.pdf

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket.

Interactions between bacteria and Candida in the burn wound

Autoregulation of the plasmid addiction operon of bacteriophage P1.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

/pdf/molecular-mechanisms-of-action-of-herbal-antifungal-alkaloid-2oyhqk53ur.pdf

Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in Candida albicans

was conducted over a period of 15 months. This study revealed that 75% of the HIV/AIDS patients had OPC. MIC testing revealed that 5% of the Candida isolates were fluconazole resistant. A correlation between CD4-T-cell counts and development of OPC in HIV/AIDS patients was also observed. Molecular typing of C. albicans isolates showed that all were genetically unrelated. Oropharyngeal candidiasis (OPC) was at the top of the list of opportunistic infections in Indian human immunodeficiency virus (HIV)/AIDS patients before the era of highly active antiretroviral therapy (1, 20, 27, 33). Although the incidences of opportunistic infection have been reduced around the globe by highly active antiretroviral therapy, the situation remains the same in most developing countries, including India, where patients can hardly afford this treatment. World Health Organization predictions have put India as one of the biggest repositories of HIV/AIDS patients in the coming decades (38, 39). In order to get an insight into the present scenario of OPC in an Indian hospital, in the present prospective study, an attempt was made to characterize the Candida species isolated from OPC over a period of 15 months in HIV/AIDS patients, mainly to know (i) the spectrum of isolates and (ii) its in vitro pattern of susceptibility to fluconazole (FLC), a common antifungal used for prophylaxis and treatment of OPC. In addition, the genetic relatedness of the isolates was also investigated to get some idea about the primary source of infection. All of the isolates included in this study were collected from 125 randomly selected HIV/AIDS patients. Whereas 100 patients showed clinical signs of OPC, 25 patients reported discomfort or pain during chewing. All of them were attending the clinic as outpatients of the All India Institute of Medical Sciences, New Delhi, India, from December 1999 to March 2001. These patients were attending the clinic for the first time and had no history of prior treatment with antifungal and antiretroviral drugs. Samples were collected with two sterile cotton swabs. One swab was used to detect the presence of any yeast by Gram staining, and the other was used to test growth on Sabouraud dextrose agar slants containing 2 g of gentamicin per ml and 0.5% cycloheximide. In the case of positive growth, yeast identification was done by conventional methods. The method used was the germ tube production test, which was done by inoculation of a single colony into 0.5 ml of horse serum, followed by incubation at 37°C for 2 h. Morphology testing for the presence of chlamydospores, pseudohyphae, true mycelium, and blastospore arrangement was done on corn meal agar. An enzymatic triphenyl tetrazolium chloride reduction test was performed in which each Candida species grows with a distinct texture and color. For further characterization, each isolate was subjected to carbohydrate assimilation and fermentation tests (2, 3). To differentiate Candida albicans from C. dubliniensis, all C. albicans isolates (confirmed by conventional methods) were subjected to growth at 45°C and a Tween 80 opacity test (13, 31). Out of 100 samples, 75 were found to be positive for Candida species infection by direct microscopy and culture. This value (75% positive) is lower than that reported (90%) in western populations (5, 10, 23, 35), and one can speculate that the Candida carriage rate in the Indian population is probably lower. Species identification revealed that C. albicans (86%) was the most predominant species; as reported earlier (20), a small percentage of other species has also been identified, i.e., C. parapsilosis (8%) and C. glabrata and C. krusei (3%). The most interesting fact is that C. tropicalis and C. dubliniensis were not isolated. This is a surprising observation in view of reports from developed countries, where non-C. albicans candidiasis is a major problem in HIV/AIDS patients (9, 11, 12, 18, 19). Antifungal drug prophylaxis is rarely practiced in India, and this may be the reason for this observation. Currently, the most widely used drug for treating candidiasis is FLC (16, 17, 24, 28, 36). In vitro FLC susceptibility testing of all of the isolates was done by microdilution susceptibility assay (22). More than 93% of the Candida isolates were found to be susceptible, with MICs of 8 g/ml; 2% were found to be dose-dependently susceptible, with MICs of 16 to 32 g/ml; and only 5% (two isolates of C. albicans and one each of C. glabrata and C. krusei) were found to be resistant, with MICs of 64 g/ml. The resistant strains (5%) were from patients who never took FLC. These resistant isolates can be described as primarily or intrinsically resistant strains. It is

https://jcm.asm.org/content/jcm/42/3/1260.full.pdf

Susceptibility Pattern and Molecular Type of Species-Specific Candida in Oropharyngeal Lesions of Indian Human Immunodeficiency Virus-Positive Patients

Gauranga Mukhopadhyay

Papers

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket.

Interactions between bacteria and Candida in the burn wound

Autoregulation of the plasmid addiction operon of bacteriophage P1.

Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in Candida albicans

Susceptibility Pattern and Molecular Type of Species-Specific Candida in Oropharyngeal Lesions of Indian Human Immunodeficiency Virus-Positive Patients