The bZIP Ap1 transcription factor is a negative regulator of virulence attributes of the anthropophilic dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a fungus that causes chronic skin and nail infections in healthy individuals and immunocompromised patients. During infection, T. rubrum invades host cutaneous tissues by adapting to the acidic pH and the innate immune response of the host. Several genes are upregulated during the growth of T. rubrum in substrates found in human tissue, including the ap1 gene, which codes for the transcription factor Ap1. Here, we generated a null mutant strain by deleting the T. rubrum ap1 gene and performed a functional analysis of this gene. Our results showed that the Δap1mutant increased its growth in nail fragments and co-cultures with keratinocytes compared to the wild type. Furthermore, the mutant displayed hyperpigmentation, thickening of the conidia cell wall, increased conidia susceptibility to calcofluor-white compared to the wild type, and loss of control of the keratinolytic activity. Although the ap1 gene was upregulated during exposure to the antifungal drugs amphotericin B, nystatin, and terbinafine, its deletion did not alter the fungal susceptibility to these drugs, revealing the role of the ap1 gene in the physiological response to the stress caused by these drugs, but not in their resistance. Moreover, ap1 was also involved in the oxidative stress response caused by menadione, but not paraquat or hydrogen peroxide. These findings indicate that the ap1 gene plays a role in the negative control of virulence-related attributes and may contribute to the chronicity of nail infection caused by T. rubrum.

StuA-Regulated Processes in the Dermatophyte Trichophyton rubrum: Transcription Profile, Cell-Cell Adhesion, and Immunomodulation.

Fungal infections represent a significant concern worldwide, contributing to human morbidity and mortality. Dermatophyte infections are among the most significant mycoses, and Trichophyton rubrum appears to be the principal causative agent. Thus, an understanding of its pathophysiology is urgently required. Several lines of evidence have demonstrated that the APSES family of transcription factors (Asm1p, Phd1p, Sok2p, Efg1p, and StuA) is an important point of vulnerability in fungal pathogens and a potential therapeutic target. These transcription factors are unique to fungi, contributing to cell differentiation and adaptation to environmental cues and virulence. It has recently been demonstrated that StuA plays a pleiotropic role in dermatophyte pathophysiology. It was suggested that it functions as a mediator of crosstalk between different pathways that ultimately contribute to adaptive responses and fungal-host interactions. The complex regulation of StuA and its interaction pathways are yet to be unveiled. Thus, this study aimed to gain a deeper understanding of StuA-regulated processes in T. rubrum by assessing global gene expression following growth on keratin or glucose sources. The data showed the involvement of StuA in biological processes related to central carbon metabolism and glycerol catabolism, reactive oxygen species metabolism, and cell wall construction. Changes in carbohydrate metabolism may be responsible for the significant alteration in cell wall pattern and consequently in cell-cell interaction and adhesion. Loss of StuA led to impaired biofilm production and promoted proinflammatory cytokine secretion in a human keratinocyte cell line. We also observed the StuA-dependent regulation of catalase genes. Altogether, these data demonstrate the multitude of regulatory targets of StuA with a critical role in central metabolism that may ultimately trigger a cascade of secondary effects with substantial impact on fungal physiology and virulence traits.

The stuA gene controls development, adaptation, stress tolerance, and virulence of the dermatophyte Trichophyton rubrum.

The APSES family, comprising of the transcriptional regulators Asm1p, Phd1p, Sok2p, Efg1p, and StuA, is found exclusively in fungi and has been reported to control several cellular processes in these organisms. However, its function in dermatophytes has not yet been completely understood. Here, we generated two null mutant strains by deleting the stuA gene in the dermatophyte Trichophyton rubrum, the most common clinical isolate obtained from human skin and nail mycoses. The functional characterization of the knocked-out strains revealed the involvement of stuA in germination, morphogenesis of conidia and hyphae, pigmentation, stress responses, and virulence. Although the mutant strains could grow under several nutritional conditions, growth on the keratin medium, human nails, and skin was impaired. The co-culture of stuA mutants with human keratinocytes revealed enhanced development. Moreover, a stuA mutant grown on the keratin substrate showed a marked decrease in the transcript numbers of the hydrophobin encoding gene (hypA), suggesting the involvement of stuA in the molecular mechanisms underlying mechanosensing during the fungi-host interaction. In addition, bioinformatics analyses revealed the potential involvement of StuA in different biological processes such as oxidation-reduction, phosphorylation, proteolysis, transcription/translation regulation, and carbohydrate metabolism. Cumulatively, the present study suggested that StuA is a crosstalk mediator of many pathways and is an integral component of the infection process, implying that it could be a potential target for antifungal therapy.

HacA Governs Virulence Traits and Adaptive Stress Responses in Trichophyton rubrum.

The ability of fungi to sense environmental stressors and appropriately respond is linked to secretory system functions. The dermatophyte infection process depends on an orchestrated signaling regulation that triggers the transcription of genes responsible for adherence and penetration of the pathogen into host-tissue. A high secretion system is activated to support the host-pathogen interaction and assures maintenance of the dermatophyte infection. The gateway of secretion machinery is the endoplasmic reticulum (ER), which is the primary site for protein folding and transport. Current studies have shown that ER stress that affects adaptive responses is primarily regulated by UPR and supports fungal pathogenicity; this has been assessed for yeasts and Aspergillus fumigatus, in regard to how these fungi cope with host environmental stressors. Fungal UPR consists of a transmembrane kinase sensor (Ire1/IreA) and a downstream target Hac1/HacA. The active form of Hac is achieved via non-spliceosomal intron removal promoted by endonuclease activity of Ire1/IreA. Here, we assessed features of HacA and its involvement in virulence and susceptibility in Trichophyton rubrum. Our results showed that exposure to antifungals and ER-stressing agents initiated the activation of HacA from T. rubrum. Interestingly, the activation occurs when a 20 nt fragment is removed from part of the exon-2 and part of intron-2, which in turn promotes the arisen of the DNA binding site motif and a dimer interface domain. Further, we found changes in the cell wall and cellular membrane composition in the ΔhacA mutant as well as an increase in susceptibility toward azole and cell wall disturbing agents. Moreover, the ΔhacA mutant presented significant defects in important virulence traits like thermotolerance and growth on keratin substrates. For instance, the development of the ΔhacA mutant was impaired in co-culture with keratinocytes or human nail fragments. Changes in the pro-inflammatory cytokine release were verified for the ΔhacA mutant during the co-culture assay, which might be related to differences in pathogen-associated molecular patterns (PAMPs) in the cell wall. Together, these results suggested that HacA is an integral part of T. rubrum physiology and virulence, implying that it is an important molecular target for antidermatophytic therapy.

Genes coding for LysM domains in the dermatophyte Trichophyton rubrum: A transcription analysis.

The filamentous fungus Trichophyton rubrum is a pathogen that causes superficial mycoses in humans, predominantly in keratinized tissues. The occurrence of dermatophytoses has increased in the last decades, mainly in immunocompromised patients, warranting research on the mechanisms involved in dermatophyte virulence. The genomes of dermatophytes are known to be enriched in genes coding for proteins containing the LysM domain, a carbohydrate-binding module, indicating the possible involvement of these genes in virulence. Although the LysM domains have already been described in other fungi, their biological functions in dermatophytes are unknown. Here we assessed the transcription of genes encoding proteins containing the LysM domains in T. rubrum grown on different substrates using quantitative real-time polymerase chain reaction. Some of these genes showed changes in transcription levels when T. rubrum was grown on keratin. In silico analyses suggest that some of these proteins share features, namely, they are anchored in the plasma membrane and contain the catalytic domain chitinase II and signal peptide domains. Here we show a detailed study of genes encoding the proteins with LysM-containing domains in T. rubrum, aiming to contribute to the understanding of their functions in dermatophytes.

Dermatophyte Resistance to Antifungal Drugs: Mechanisms and Prospectus.

Dermatophytes comprise pathogenic fungi that have a high affinity for the keratinized structures present in nails, skin, and hair, causing superficial infections known as dermatophytosis. A reasonable number of antifungal drugs currently exist on the pharmaceutical market to control mycoses; however, their cellular targets are restricted, and fungi may exhibit tolerance or resistance to these agents. For example, the stress caused by antifungal and cytotoxic drugs in sub-inhibitory concentrations promotes compensatory stress responses, with the over-expression of genes involved in cellular detoxification, drug efflux, and signaling pathways being among the various mechanisms that may contribute to drug tolerance. In addition, the ATP-binding cassette transporters in dermatophytes that are responsible for cellular efflux can act synergistically, allowing one to compensate for the absence of the other, revealing the complexity of drug tolerance phenomena. Moreover, mutations in genes coding for target enzymes could lead to substitutions in amino acids involved in the binding of antifungal agents, hindering their performance and leading to treatment failure. The relevance of each one of these mechanisms of resistance to fungal survival is hard to define, mainly because they can act simultaneously in the cell. However, an understanding of the molecular mechanisms involved in the resistance/tolerance processes, the identification of new antifungal targets, as well as the prospective of new antifungal compounds among natural or synthetic products, are expected to bring advances and new insights that facilitate the improvement or development of novel strategies for antifungal therapy.

Terbinafine resistance conferred by multiple copies of the salicylate 1-monooxygenase gene in Trichophyton rubrum.

Resistance to antifungals is a leading concern in the treatment of human mycoses. We demonstrate that the salA gene, encoding salicylate 1-monooxygenase, is involved in resistance of the dermatophyte Trichophyton rubrum to terbinafine, one of the most effective antifungal drugs against dermatophytes. A strain with multiple copies of salA was constructed and exhibited elevated expression of salA and increased terbinafine resistance. This reflects a mechanism not yet reported in a pathogenic fungus.

Heat Shock Proteins in Dermatophytes: Current Advances and Perspectives.

Heat shock proteins (HSPs) are proteins whose transcription responds rapidly to temperature shifts. They constitute a family of molecular chaperones, involved in the proper folding and stabilisation of proteins under physiological and adverse conditions. HSPs also assist in the protection and recovery of cells exposed to a variety of stressful conditions, including heat. The role of HSPs extends beyond chaperoning proteins, as they also participate in diverse cellular functions, such as the assembly of macromolecular complexes, protein transport and sorting, dissociation of denatured protein aggregates, cell cycle control, and programmed cell death. They are also important antigens from a variety of pathogens, are able to stimulate innate immune cells, and are implicated in acquired immunity. In fungi, HSPs have been implicated in virulence, dimorphic transition, and drug resistance. Some HSPs are potential targets for therapeutic strategies. In this review, we discuss the current understanding of HSPs in dermatophytes, which are a group of keratinophilic fungi responsible for superficial mycoses in humans and animals. Computational analyses were performed to characterise the group of proteins in these dermatophytes, as well as to assess their conservation and to identify DNA-binding domains (5'-nGAAn-3') in the promoter regions of the hsp genes. In addition, the quantification of the transcript levels of few genes in a pacC background helped in the development of an extended model for the regulation of the expression of the hsp genes, which supports the participation of the pH-responsive transcriptional regulator PacC in this process.

Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum.

Treatment of fungal infections is difficult due to several reasons, such as side effects of drugs, emergence of resistant strains, and limited number of molecular targets for the drug compounds. In fungi, heat shock proteins (Hsps) have been implicated in several processes with the conserved molecular chaperone Hsp90 emerging as a potential target for antifungal therapy. It plays key cellular roles by eliciting molecular response to environmental changes, morphogenesis, antifungal resistance, and fungal pathogenicity. Here, we evaluated the transcription profiles of hsp genes of the most prevalent dermatophyte Trichophyton rubrum in response to different environmental challenges including nutrient availability, interaction with cells and molecules of the host tissue, and drug exposure. The results suggest that each Hsp responds to a specific stress condition and that the cohort of Hsps facilitates fungal survival under various environmental challenges. Chemical inhibition of Hsp90 resulted in increased susceptibility of the fungus to itraconazole and micafungin, and decreased its growth in human nails in vitro. Moreover, some hsp and related genes were modulated by Hsp90 at the transcriptional level. We are suggesting a role of Hsp90 in the pathogenicity and drug susceptibility of T. rubrum as well as the regulation of other Hsps. The synergism observed between the inhibition of Hsp90 and the effect of itraconazole or micafungin in reducing the fungal growth is of great interest as a novel and potential strategy to treat dermatophytoses.

Implication of the VirD4 coupling protein of the Lvh type 4 secretion system in virulence phenotypes of Legionella pneumophila.

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm(+), showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm(+) background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.

Cold shock genes cspA and cspB from Caulobacter crescentus are posttranscriptionally regulated and important for cold adaptation.

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

CspC and CspD are essential for Caulobacter crescentus stationary phase survival.

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance.

Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting <10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.

Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain.

The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30(per cente) of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function. (au)