A one-pot five component reaction for the synthesis of tetrazol-benzofuran hybrids and their inhibitory activity against Mucor lusitanicus.

A synthetic strategy for obtaining a new series of 1,5-disubstituted tetrazole-benzofuran hybrid systems via a one-pot five-component reaction is described. This process involves a Ugi-azide multicomponent reaction coupled to an intramolecular cyclization catalyzed by Pd/Cu, resulting in low to moderate yields from 21 to 67%. This protocol allowed the synthesis of highly substituted benzofurans at the 2-position through an operationally simple process under mild reaction conditions and with high bond forming efficiency due to the formation of six new bonds (two C-C, two C-N, one N-N, and one C-O). Besides, to evaluate the antifungal activity of 1,5-disubstituted tetrazole-benzofurans 9a-n, in vitro studies against Mucor lusitanicus were performed, finding that compound 9b exhibits bioactivity comparable to the commercial antifungal drug Amphotericin B. These results suggest potential for use in controlling mucormycosis infections in animal models, highlighting the importance of these findings given the limited antifungal drug options and high mortality rates associated with this infection.

Transcription Factors Tec1 and Tec2 Play Key Roles in the Hyphal Growth and Virulence of Mucor lusitanicus Through Increased Mitochondrial Oxidative Metabolism.

Mucormycosis is a lethal and difficult-to-treat fungal infection caused by fungi of the order Mucorales. Mucor lusitanicus, a member of Mucorales, is commonly used as a model to understand disease pathogenesis. However, transcriptional control of hyphal growth and virulence in Mucorales is poorly understood. This study aimed to investigate the role of Tec proteins, which belong to the TEA/ATTS transcription factor family, in the hyphal development and virulence of M. lusitanicus. Unlike in the genome of Ascomycetes and Basidiomycetes, which have a single Tec homologue, in the genome of Mucorales, two Tec homologues, Tec1 and Tec2, were found, except in that of Phycomyces blakesleeanus, with only one Tec homologue. tec1 and tec2 overexpression in M. lusitanicus increased mycelial growth, mitochondrial content and activity, expression of the rhizoferrin synthetase-encoding gene rfs, and virulence in nematodes and wax moth larvae but decreased cAMP levels and protein kinase A (PKA) activity. Furthermore, tec1- and tec2-overexpressing strains required adequate mitochondrial metabolism to promote the virulent phenotype. The heterotrimeric G beta subunit 1-encoding gene deletant strain (Δgpb1) increased cAMP-PKA activity, downregulation of both tec genes, decreased both virulence and hyphal development, but tec1 and tec2 overexpression restored these defects. Overexpression of allele-mutated variants of Tec1(S332A) and Tec2(S168A) in the putative phosphorylation sites for PKA increased both virulence and hyphal growth of Δgpb1. These findings suggest that Tec homologues promote mycelial development and virulence by enhancing mitochondrial metabolism and rhizoferrin accumulation, providing new information for the rational control of the virulent phenotype of M. lusitanicus.

Blood Serum Stimulates the Virulence Potential of Mucorales through Enhancement in Mitochondrial Oxidative Metabolism and Rhizoferrin Production.

This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.

Volatile Fingerprint Mediates Yeast-to-Mycelial Conversion in Two Strains of Beauveria bassiana Exhibiting Varied Virulence.

Beauveria bassiana is a dimorphic and entomopathogenic fungus with different ecological roles in nature. In pathogenic fungi, yeast-to-mycelial conversion, which is controlled by environmental factors, is required for virulence. Here, we studied the effects of different stimuli on the morphology of two B. bassiana strains and compared the toxicities of culture filtrates. In addition, we explored the role of volatiles as quorum sensing-like signals during dimorphic transition. The killing assays in Caenorhabditis elegans (Nematoda: Rhabditidae) showed that strain AI2 isolated from a mycosed insect cadaver had higher toxicity than strain AS5 isolated from soil. Furthermore, AI2 showed earlier yeast-to-mycelial switching than AS5. However, an increase in inoculum size induced faster yeast-to-mycelium conversion in AS5 cells, suggesting a cell-density-dependent phenomenon. Gas chromatography-mass spectrometry (GC-MS) analyses showed that the fingerprint of the volatiles was strain-specific; however, during the morphological switching, an inverse relationship between the abundance of total terpenes and 3-methylbutanol was observed in both strains. Fungal exposure to 3-methylbutanol retarded the yeast-to-mycelium transition. Hence, this study provides evidence that volatile compounds are associated with critical events in the life cycle of B. bassiana.

Fungal Alcohol Dehydrogenases: Physiological Function, Molecular Properties, Regulation of Their Production, and Biotechnological Potential.

Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.

Metformin, a biological and synthetic overview.

Metformin is the most widely known anti-hyperglycemic, officially acquired by the USA government in 1995 and in 2001 it became the most prescribed treatment for type II diabetes. But how did it become the must-use drug for this disease in such a short period of time? it all started with traditional medicine, by using a plant known as "goat's rue" for the reduction of blood glucose levels. Its use arose in 1918 and evolved to the metformin synthesis in laboratories a couple of years later, using very rudimentary methods which involved melting and strong heating. Thus, a first synthetic route that allowed the preparation of the initial metformin derivates was established. Some of these resulted toxics, and others outperformed the metformin, reducing the blood glucose levels in such efficient way. Nevertheless, the risk and documented cases of lactic acidosis increased with metformin derivatives like buformin and phenformin. Recently, metformin has been widely studied, and it has been associated and tested in the treatment of type II diabetes, cancer, polycystic ovarian syndrome, cell differentiation to oligodendrocytes, reduction of oxidative stress in cells, weight reduction, as anti-inflammatory and even in the recent COVID-19 disease. Herein we briefly review and analyze the history, synthesis, and biological applications of metformin and its derivates.

Role of the nonribosomal peptide synthetase siderophore enzyme (Rfs) of Mucor lusitanicus in controlling the growth of fungal phytopathogens.

Dimorphic species of Mucor, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of Mucor are sources of biotechnological products, such as biodiesel from Mucor circinelloides and expression of heterologous proteins from Mucor lusitanicus. Furthermore, Mucor lusitanicus has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between Mucor lusitanicus and other soil inhabitants. In this study, we investigated the potential use of Mucor lusitanicus as a biocontrol agent against fungal phytopathogens, namely Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and Alternaria solani, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from Mucor lusitanicus inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of Mucor lusitanicus carrying the deletion of rfs gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an rfs-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from Mucor lusitanicus in the biocontrol of fungal phytopathogens.

Genome mining, phylogenetic, and functional analysis of arsenic (As) resistance operons in Bacillus strains, isolated from As-rich hot spring microbial mats.

The geothermal zone of Araró, México, is located within the trans-Mexican volcanic belt, an area with numerous arsenic (As)-rich hot springs. In this study, the draft genome sequence of two endemic Bacillus strains (ZAP17 and ZAP62) from Araró microbial mat hot springs was determined, which were able to grow on arsenate As(V) (up to 64 mM) and arsenite As(III) (up to 32 mM). Phylogenetic analysis based on 16 S rRNA and gyrB sequences, as well as genome sequence analysis based on average nucleotide identity (>96 %) and digital DNA-DNA hybridization (>70 %), indicated that these strains belong to the Bacillus paralicheniformis ZAP17 and Bacillus altitudinis ZAP62. Furthermore, through genome mining, it was identified two arsenic resistance operons, arsRBC, and arsRBCDA in both strains as potential determinants of As resistance. Predicted ArsA (arsenial pump-driving ATPase), ArsB (Arsenical efflux pump protein), ArsC (Arsenate reductase), ArsD (Arsenical efflux pump protein) and ArsR (Metalloregulator/ars operon repressor) proteins, clearly grouped with their respective clades corresponding to other characterized bacterial species, mainly Firmicutes. To further evaluate the functionality of the ars operons in ZAP17 and ZAP62 strains, our results showed that arsRBC and arsRBCDA genes were expressed in the presence of As(III). Finally, the presence of ars operons in the genome of Bacillus species residing in As-rich environments, such as the Araró hot springs, might be a potential mechanism to survive under such harsh conditions.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus.

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Novel 2-aryl-4-aryloxyquinoline-based fungistatics for Mucor circinelloides. Biological evaluation of activity, QSAR and docking study.

Zygomycetes are ubiquitous saprophytes in natural environments which transform organic matter. Some zygomycetes of gender Mucor have attracted interest in health sector. Due to its ability as opportunistic microorganisms infecting immuno-compromised people and to the few available pharmacological treatments, the mucormycosis is receiving worldwide attention. Concerning to the pharmacological treatments, some triazole-based compounds such as fluconazole are extensively used. Nevertheless, we focused in the quinolines since they are broadly used models for the design and development of new synthetic antifungal agents. In this study, the fungistatic activity on M. circinelloides of various 2-aryl-4-aryloxyquinoline-based compounds was discovered, and in some cases, it resulted better than reference compound fluconazole. These quinoline derivatives were synthesized via the Csp2-O bond formation using diaryliodonium(III) salts chemistry. A QSAR study was carried out to quantitatively correlate the chemical structure of the tested compounds with their biological activity. Also, a docking study to identify a plausible action target of our more active quinolines was carried out. The results highlighted an increased activity with the fluorine- and nitro-containing derivatives. In light of the few mucormycosis pharmacological treatments, herein we present some non-described molecules with excellent in vitro activities and potential use in the mucormycosis treatment.

Expression of bik cluster and production of bikaverin by Fusarium oxysporum f. sp. lycopersici grown using two alternate nitrogen sources.

The genus Fusarium can be utilized to produce a great variety of secondary metabolites under specific culture conditions, including pigments of increasing biotechnological interest, such as bikaverin. Such pigments are important due to the biological properties they possess, including antitumor and antibiotic activities, among others. In Fusarium fujikuroi, bik1-bik6 have been identified as the genes that are responsible for the synthesis of bikaverin. Therefore, in this study, we screened for the presence of bik genes and examined changes in mRNA levels of the bik genes under the influence of NH4NO3 (0.024, 0.048, 0.50, 1.0, and 4.60 g L-1) and NH4Cl (0.50 and 1.0 g L-1) as nitrogen sources for the phytopathogen Fusarium oxysporum f. sp. lycopersici. Our results indicated the presence of at least six bik (bik1-bik6) genes and showed increased mRNA levels for bik4, bik5, and bik6 in conditions where NH4NO3 was used at pH 3.0. The characteristic coloration of bikaverin was obtained in 10 out of 16 culture conditions, except when the fungus was grown with higher concentrations of NH4NO3 (1.0 and 4.60 g L-1). The pigment was chloroform-extracted from the culture conditions of NH4NO3 (0.024, 0.048, and 0.50 g L-1) and NH4Cl (0.50 and 1.0 g L-1) with 3 and 9 days of incubation. Analysis via visible spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry were used for the identification of bikaverin.

Transcript pattern analysis of Arf-family genes in the phytopathogen Fusarium oxysporum f. sp. lycopersici reveals the role of Arl3 in the virulence.

Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as a critical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.

The heterotrimeric G-protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides.

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G-protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1-encoding gene is up-regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild-type strain. Mutation in a regulatory subunit of cAMP-dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild-type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.

Virulence Conferred by PumA Toxin from the Plasmid-Encoded PumAB Toxin-Antitoxin System is Regulated by Quorum System.

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin that are important for plasmid stabilization (plasmid-encoded) and bacterial virulence (chromosome-encoded). These systems are also related to biofilm and persister cell formations. Pseudomonas aeruginosa is an antibiotic-resistant human pathogen that produces virulence factors modulated by quorum sensing (QS) and can form biofilms. The type II PumAB TA system of pUM505, isolated from a clinical strain of P. aeruginosa, confers plasmid stability. Additionally, the PumA toxin increases P. aeruginosa virulence and is neutralized by the PumB antitoxin. In this study, we determined whether virulence conferred by PumA toxin is regulated by QS. The pumA gene was transferred to P. aeruginosa lasI/rhlI, a mutant strain in the LasI and RhlI QS systems, to analyze the effect on virulence of the transformants. pumA transfer did not increase bacterial virulence in lettuce and Caenorhabditis elegans, suggesting that the virulence conferred by PumA requires QS modulation. pumA mRNA levels drastically decreased in the P. aeruginosa lasI/rhlI (pUC_pumA) strain, suggesting positive regulation of pumA gene expression by QS. Supplementation of the growth medium of P. aeruginosa lasI/rhlI (pUC_pumA) with C4-AHL and 3-oxo-C12-AHL autoinducers increased pumA mRNA levels and restored bacterial virulence, suggesting that both autoinducers complemented the mutations and positively regulated the toxic effects of PumA. This strengthened the hypothesis that QS regulates bacterial virulence conferred by the PumA toxin. Thus, this report establishes an important function of QS in the virulence conferred by plasmid-encoded TA systems in bacterial pathogens.

Arf-like proteins (Arl1 and Arl2) are involved in mitochondrial homeostasis in Mucor circinelloides.

Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(-) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(-) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose. To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.

Sporulation on blood serum increases the virulence of Mucor circinelloides.

Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1ß, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

Role of Arf-like proteins (Arl1 and Arl2) of Mucor circinelloides in virulence and antifungal susceptibility.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals.

OBJECTIVES: This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. METHODS: A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. RESULTS: The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. CONCLUSIONS: These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

A plasmid-encoded mobile genetic element from Pseudomonas aeruginosa that confers heavy metal resistance and virulence.

Mobile plasmid-encoded elements are DNA segments that are transferred for horizontal gene transfer and that confer adaptive proprieties, as well as virulence and antibiotic and heavy metal resistance to bacteria. The conjugative plasmid pUM505, isolated from a clinical strain of Pseudomonas aeruginosa, possesses a putative 31.292 kb mobile element (denominated Mpe: Mobile plasmid- encoded element) that, in addition to possessing chr genes that confer chromate resistance to Pseudomonas, contains two putative mer operons that could confer mercury resistance. Moreover, the Mpe contains genes related previously with the virulence of both P. aeruginosa and Escherichia coli strains. In this work, we determined that Mpe from pUM505 was able to independently move to another DNA molecule, conferring chromate and mercury resistance to P. aeruginosa PAO1 and mercury resistance to E. coli JM101, suggesting that its transference might be beneficial to bacteria under certain environmental conditions. Additionally, the transference of Mpe increased the virulence of P. aeruginosa PAO1 against the nematode Caenorhabditis elegans, suggesting its contribution to the pathogenicity of P. aeruginosa. In this work, we describe a new mobile plasmid-encoded element that possesses the potential to be transferred by horizontal gene transference, which could provide bacteria with a wide variety of adaptive traits such as heavy metal resistance and virulence, which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.