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To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.

It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.

Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.

Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history. Most ancient ‘primary’ endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome) 1 . Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into ‘mucus-filled’ spheres, which surround the host cell nucleus and occupy most of the cytoplasm 2 . The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles. Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells 3 , 4 , and that bacteria within spheres comprise these two species 5 . Here we show that mealybug host cells do indeed harbour both β- and γ-subdivision Proteobacteria, but they are not co-inhabitants of the spheres. Rather, we show that the symbiotic spheres themselves are β-proteobacterial cells. Thus, γ-Proteobacteria live symbiotically inside β-Proteobacteria. This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.

Pulmonary infections with Mycobacterium avium subs. Hominissuis occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. M. avium is able to survive and grow in many conditions, including water distribution systems and residential plumbing fixtures, placing the opportunistic pathogen in frequent proximity to humans. Despite the worldwide distribution and increasing incidence of M. avium pulmonary disease, understanding of M. avium biofilms, their development, constituent cell phenotypes, and implications of biofilm growth on subsequent pathogenesis is limited.One source of ambiguity in our understanding of M. avium biofilms is the use of different in vitro models to characterize biofilm development in different studies, leading to uncertainty about whether findings are characteristic of M. avium biofilms in general or whether they arise from conditions specific to a given model. To begin elucidating the similarities and differences between biofilms formed using different methods, we made a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and drug susceptibility of biofilm resident bacteria, using two published methods to generate M. avium biofilms: four week incubation in M63 medium or 24 hour exposure to dithiothreitol (DTT). Differences between the biofilms were observed in ultrastructure appearances visualized by scanning electron microscopy and in the susceptibility of the biofilms to degradation by enzymes targeting ECM components. However, staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Further, bacteria in both biofilms displayed resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at >10-fold lower rates than planktonic cells at almost all concentrations tested.To better understand transcriptional adaptations that occur in M. avium during residence in a biofilm and how those expression patterns change following engulfment by macrophages, we undertook the first study of global differential gene expression in M. avium using RNAseq. This analysis revealed largely divergent transcriptional profiles in M. avium taken from planktonic culture and grown in M63 or DTT biofilms. However, M63 biofilm M. avium and planktonic M. avium used to infect RAW264.7 macrophages for 24 hours had similar transcriptional responses to infection. Comparison of genes that were differentially expressed in both planktonic cells post infection and in M63 biofilm derived cells but were not differentially expressed in M63 cells post infection suggested that gene expression patterns induced by the M63 biofilm environment might prepare M. avium to persist during infection. To examine this possibility further, we carried out aerosol infections of BALB/c mice with M. avium grown either in planktonic culture or in an M63 biofilm. Regardless of the M. avium growth condition, the bacterial loads in the lungs and tracheae of the mice progressively decreased over a 21 day infection period, revealing no apparent difference in survival ability between biofilm and planktonic M. avium under the conditions tested.Taken together, the findings presented in this dissertation indicate that the mechanism of biofilm formation in M. avium, including patterns of differential gene expression, varies under different environmental conditions. However, our analyses of the M63 and DTT biofilm models suggest that some characteristics may be common to M. avium biofilms, including the production and export of cellulose, increased antibiotic resistance, and increased expression of certain genes, such as those involved in stress responses. While growth in a biofilm may cause patterns of expression in some genes that are similar to those observed during infection of macrophages, we did not observe a clear benefit for biofilm derived cells in establishing and persisting in the lungs or tracheae in our mouse aerosol infection model.

Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history. Most ancient 'primary' endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome). Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into 'mucus-filled' spheres, which surround the host cell nucleus and occupy most of the cytoplasm. The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles. Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells, and that bacteria within spheres comprise these two species. Here we show that mealybug host cells do indeed harbour both b- and g-subdivision Proteobacteria, but they are not co-inhabitants of the spheres. Rather, we show that the symbiotic spheres themselves are b-proteobacterial cells. Thus, g-Proteobacteria live symbiotically inside b-Proteobacteria. This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.