Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients.

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.

Mutation on lysX from Mycobacterium avium hominissuis impacts the host-pathogen interaction and virulence phenotype.

The lysX gene from Mycobacterium avium hominissuis (MAH) is not only involved in cationic antimicrobial resistance but also regulates metabolic activity. An MAH lysX deficient mutant was shown to exhibit a metabolic shift at the extracellular state preadapting the bacteria to the conditions inside host-cells. It further showed stronger growth in human monocytes. In the present study, the LysX activity on host-pathogen interactions were analyzed. The lysX mutant from MAH proved to be more sensitive toward host-mediated stresses such as reactive oxygen species. Further, the lysX mutant exhibited increased inflammatory response in PBMC and multinucleated giant cell (MGC) formation in human macrophages during infection studies. Coincidentally, the lysX mutant strain revealed to be more reproductive in the Galleria mellonella infection model. Together, these data demonstrate that LysX plays a role in regulating the bacillary load in host organisms and the lack of lysX gene facilitates MAH adaptation to intracellular host-habitat, thereby suggesting an essential role of LysX in the modulation of host-pathogen interaction.

Identification of cross-reactive antibodies for the detection of lymphocytes, myeloid cells and haematopoietic precursors in the naked mole rat.

The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Cytopathogenicity of Balamuthia mandrillaris, an opportunistic causative agent of granulomatous amebic encephalitis.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time- and dose-related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact-dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.