Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans.

Pathogenic microbes are exposed to a number of potential DNA-damaging stimuli during interaction with the host immune system. Microbial survival in this situation depends on a fine balance between the maintenance of DNA integrity and the adaptability provided by mutations. In this study, we investigated the association of the DNA repair response with the virulence of Cryptococcus neoformans, a basidiomycete that causes life-threatening meningoencephalitis in immunocompromised individuals. We focused on the characterization of C. neoformansAPN1 and APN2 putative genes, aiming to evaluate a possible role of the predicted Apurinic/apyrimidinic (AP) endonucleases 1 and 2 of the base excision repair (BER) pathway on C. neoformans response to stress conditions and virulence. Our results demonstrated the involvement of the putative AP-endonucleases Apn1 and Apn2 in the cellular response to DNA damage induced by alkylation and by UV radiation, in melanin production, in tolerance to drugs and in virulence of C. neoformans in vivo. We also pointed out the potential use of DNA repair inhibitor methoxy-amine in combination with conventional antifungal drugs, for the development of new therapeutic approaches against this human fungal pathogen. This work provides new information about the DNA damage response of the highly important pathogenic fungus C. neoformans.

Transcriptional Remodeling Patterns in Murine Dendritic Cells Infected with Paracoccidioides brasiliensis: More Is Not Necessarily Better.

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway's repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model.

A hidden battle in the dirt: Soil amoebae interactions with Paracoccidioides spp.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.

[Determination of single nucleotide mutations in the 23S rRNA gene of Helicobacter pylori related to clarithromycin resistance in a population from Cauca, Colombia]. / Determinación de mutaciones de un solo nucleótido en el gen 23S rRNA de Helicobacter pylori relacionadas con resistencia a claritromicina en una población del departamento del Cauca, Colombia.

INTRODUCTION: Antibiotic combination therapy for the eradication of Helicobacter pylori should be based on local resistance patterns. OBJECTIVE: To d etermine the resistance of H. pylori to clarithromycin in a population from Cauca province, through the identification of mutations in the 23S rRNA gene in DNA from gastric biopsies. MATERIALS AND METHODS: A total of 162 patients with functional dyspepsia were included in the study. The 23S rRNA gene and the DNA from 162 gastric specimens were amplified by PCR, and the mutation pattern was identified by direct sequencing. RESULTS: The frequency of clarithromycin resistance was 4%. A2143G mutation was found in four patients (2.46%) and A2142G mutation was found in three patients (1.85%). CONCLUSIONS: Our study shows that the most frequent genotype in H. pylori -positive specimens was A2143G, followed by A2142G. The observed resistance prevalence of H. pylori was low; thus, we consider that clarithromycin treatment is a valid option for H. pylori eradication in the study population.

Determinación de mutaciones de un solo nucleótido en el gen 23S rRNA de Helicobacter pylori relacionadas con resistencia a claritromicina en una población del departamento del Cauca, Colombia / Determination of single nucleotide mutations in the 23S rRNA gene of Helicobacter pylori related to clarithromycin resistance in a population from Cauca, Colombia

Introducción. La terapia antibiótica combinada para la erradicación de Helicobacter pylori debería basarse en los patrones locales de resistencia. Objetivo. Determinar la resistencia de H. pylori a claritromicina en una población del departamento del Cauca mediante la identificación de mutaciones en el gen 23S r RNA en ADN obtenido de biopsias gástricas. Materiales y métodos. Se incluyeron en el estudio 162 pacientes con dispepsia funcional. El gen 23S rRNA se amplificó por PCR y el patrón de mutaciones se identificó por secuenciación directa. Resultados. La frecuencia de resistencia a claritromicina fue de 4 %. La mutación A2143G del gen se encontró en cuatro pacientes (2,46 %) y la mutación A2142G, en tres pacientes (1,85 %). Conclusiones. El estudio encontró que el genotipo más frecuente en los especímenes positivos para H. pylori fue 2143G, seguido por A2142G. La prevalencia observada de resistencia de H. pylori fue baja; por lo tanto, se considera que el tratamiento con claritromicina es una opción válida para la erradicación de H. pylori en la población objeto de estudio.

Introduction: Antibiotic combination therapy for the eradication of Helicobacter pylori should be based on local resistance patterns. Objective: To d etermine the resistance of H. pylori to clarithromycin in a population from Cauca province, through the identification of mutations in the 23S rRNA gene in DNA from gastric biopsies. Materials and methods: A total of 162 patients with functional dyspepsia were included in the study. The 23S rRNA gene and the DNA from 162 gastric specimens were amplified by PCR, and the mutation pattern was identified by direct sequencing. Results: The frequency of clarithromycin resistance was 4%. A2143G mutation was found in four patients (2.46%) and A2142G mutation was found in three patients (1.85%). Conclusions: Our study shows that the most frequent genotype in H. pylori -positive specimens was A2143G, followed by A2142G. The observed resistance prevalence of H. pylori was low; thus, we consider that clarithromycin treatment is a valid option for H. pylori eradication in the study population.