Host stress drives tolerance and persistence: The bane of anti-microbial therapeutics.

Antibiotic resistance, typically associated with genetic changes within a bacterial population, is a frequent contributor to antibiotic treatment failures. Antibiotic persistence and tolerance, which we collectively term recalcitrance, represent transient phenotypic changes in the bacterial population that prolong survival in the presence of typically lethal concentrations of antibiotics. Antibiotic recalcitrance is challenging to detect and investigate-traditionally studied under in vitro conditions, our understanding during infection and its contribution to antibiotic failure is limited. Recently, significant progress has been made in the study of antibiotic-recalcitrant populations in pathogenic species, including Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica, and Yersiniae, in the context of the host environment. Despite the diversity of these pathogens and infection models, shared signals and responses promote recalcitrance, and common features and vulnerabilities of persisters and tolerant bacteria have emerged. These will be discussed here, along with progress toward developing therapeutic interventions to better treat recalcitrant pathogens.

Genome-wide screen of Mycobacterium tuberculosis- infected macrophages identified the GID/CTLH complex as a determinant of intracellular bacterial growth.

The eukaryotic GID/CTLH complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host antimicrobial defenses has not been described. We exploited Mycobacterium tuberculosis ( Mtb ) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 ( GID8 , YPEL5 , WDR26 , UBE2H , MAEA ) of the 10 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the antimicrobial properties of the GID/CTLH complex knockdown macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to cell death. Meanwhile, Mtb isolated from GID/CTLH knockdown macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host antimicrobial responses against intracellular bacterial infections.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

HIV-1 active and latent infections induce disparate chromatin reorganization and transcriptional regulation of mRNAs and lncRNAs in SupT1 cells.

IMPORTANCE: HIV-1 infection of T-lymphocytes depends on co-opting cellular transcriptional and translational machineries for viral replication. This requires significant changes in the cellular microenvironment. We have characterized and compared the changes in cellular chromatin structures as well as gene expression landscapes in T cells that are either actively or latently infected with HIV-1. Our results reveal that chromatin accessibility and expression of both protein-coding mRNAs and non-coding lncRNAs are uniquely regulated in HIV-1-infected T cells, depending on whether the virus is actively transcribing or remains in a transcriptionally silent, latent state. HIV-1 latent infection elicits more robust changes in the cellular chromatin organization than active viral infection. Our analysis also identifies the effects of such epigenomic changes on the cellular gene expression and subsequent biological pathways. This study comprehensively characterizes the cellular epigenomic and transcriptomic states that support active and latent HIV-1 infection in an in vitro model of SupT1 cells.

Investigation into the Mechanism of Action of the Tuberculosis Drug Candidate SQ109 and Its Metabolites and Analogues in Mycobacteria.

We tested a series of SQ109 analogues against Mycobacterium tuberculosis and M. smegmatis, in addition to determining their uncoupling activity. We then investigated potential protein targets, involved in quinone and cell wall biosynthesis, using "rescue" experiments. There was little effect of menaquinone on growth inhibition by SQ109, but there were large increases in the IC50 of SQ109 and its analogues (up to 20×) on addition of undecaprenyl phosphate (Up), a homologue of the mycobacterial decaprenyl (C50) diphosphate. Inhibition of an undecaprenyl diphosphate phosphatase, an ortholog of the mycobacterial phosphatase, correlated with cell growth inhibition, and we found that M. smegmatis cell growth inhibition could be well predicted by using uncoupler and Up-rescue results. We also investigated whether SQ109 was metabolized inside Mycobacterium tuberculosis, finding only a single metabolite, previously shown to be inactive. The results are of general interest since they help explain the mechanism of SQ109 in mycobacteria.

The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Protocol for multi-modal single-cell RNA sequencing on M. tuberculosis-infected mouse lungs.

To elucidate how different immune cells contribute to control or progression of M. tuberculosis (Mtb) infection, we developed a technique to perform multi-modal single-cell RNA sequencing (scRNA-seq) from in vivo Mtb-infected lung macrophages. This protocol simultaneously acquires the transcriptome, surface marker expression, and bacterial phenotype of each infected cell. We describe steps for sorting Mtb-infected cells and staining with CITE-seq antibodies, as well as for methanol fixation and generation of scRNA-seq libraries. This protocol can be used on tissues derived from murine, nonhuman primate, and human infections. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2021).1.

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism.

Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), contributes to approximately 1.5 million deaths every year. Prior work has established that lipids are actively catabolized by Mtb in vivo and fulfill major roles in Mtb physiology and pathogenesis. We conducted a high-throughput screen to identify inhibitors of Mtb survival in its host macrophage. One of the hit compounds identified in this screen, sAEL057, demonstrates highest activity on Mtb growth in conditions where cholesterol was the primary carbon source. Transcriptional and functional data indicate that sAEL057 limits Mtb's access to iron by acting as an iron chelator. Furthermore, pharmacological and genetic inhibition of iron acquisition results in dysregulation of cholesterol catabolism, revealing a previously unappreciated linkage between these pathways. Characterization of sAEL057's mode of action argues that Mtb's metabolic regulation reveals vulnerabilities in those pathways that impact central carbon metabolism.

Nutrition, Inflammation, and the Gut Microbiota among Outpatients with Active Tuberculosis Disease in India.

India has the highest rates of tuberculosis (TB) globally and a high prevalence of malnutrition; however, the interplay between host nutritional status, inflammation, and the gut microbiome in active tuberculosis disease (ATBD) is less well-studied. We examined differences in gut microbial composition and diversity based on undernutrition and inflammation status among outpatients with ATBD at the time of treatment initiation. During this exploratory cross-sectional study, outpatients (N = 32) with ATBD (confirmed by Xpert MTB/RIF) were enrolled in anti-TB treatment initiated at a hospital in rural southern India. The 16S rRNA sequencing was used to assess the composition of the gut microbiome. We assessed multiple markers of nutritional status, including micronutrient status concentrations (vitamin D [25(OH)D], vitamin B12, ferritin), anthropometry (body mass index, mid-upper arm circumference, and height), and C-reactive protein (CRP), as indicators of inflammation. We found that 25(OH)D was positively associated with the relative abundance of Oscillospira spp., a butyrate-producing genus linked with anti-inflammation effects, and that ferritin was positively associated with Proteobacteria taxa, which have been associated with worse inflammation in other studies. Finally, we found a greater abundance of inflammation-associated taxa from the Proteobacteria phylum and lower alpha-diversity indices among those who were underweight or who had low mid-upper arm circumference or short stature. In summary, we found differences in the gut microbiota composition and diversity among those with undernutrition compared with those with adequate nutrition status at the time of initiation of treatment among patients with ATBD in India. Clinical implications of these findings will need to be examined by larger longitudinal studies.

In Vitro Miniaturized Tuberculosis Spheroid Model.

Tuberculosis (TB) is a public health concern that impacts 10 million people around the world. Current in vitro models are low throughput and/or lack caseation, which impairs drug effectiveness in humans. Here, we report the generation of THP-1 human monocyte/macrophage spheroids housing mycobacteria (TB spheroids). These TB spheroids have a central core of dead cells co-localized with mycobacteria and are hypoxic. TB spheroids exhibit higher levels of pro-inflammatory factor TNFα and growth factors G-CSF and VEGF when compared to non-infected control. TB spheroids show high levels of lipid deposition, characterized by MALDI mass spectrometry imaging. TB spheroids infected with strains of differential virulence, Mycobacterium tuberculosis (Mtb) HN878 and CDC1551 vary in response to Isoniazid and Rifampicin. Finally, we adapt the spheroid model to form peripheral blood mononuclear cells (PBMCs) and lung fibroblasts (NHLF) 3D co-cultures. These results pave the way for the development of new strategies for disease modeling and therapeutic discovery.

Parental Enrollment Decision-Making for a Neonatal Clinical Trial.

OBJECTIVE: To describe the parental experience of recruitment and assess differences between parents who participated and those who declined to enroll in a neonatal clinical trial. STUDY DESIGN: This was a survey conducted at 12 US neonatal intensive care units of parents of infants who enrolled in the High-dose Erythropoietin for Asphyxia and encephaLopathy (HEAL) trial or who were eligible but declined enrollment. Questions assessed 6 factors of the parental experience of recruitment: (1) interactions with research staff; (2) the consent experience; (3) perceptions of the study; (4) decisional conflict; (5) reasons for/against participation; and (6) timing of making the enrollment decision. RESULTS: In total, 269 of 387 eligible parents, including 183 of 242 (75.6%) of those who enrolled their children in HEAL and 86 of 145 (59.3%) parents who declined to enroll their children in HEAL, were included in analysis. Parents who declined to enroll more preferred to be approached by clinical team members rather than by research team members (72.9% vs 49.2%, P = .005). Enrolled parents more frequently reported positive initial impressions (54.9% vs 10.5%, P < .001). Many parents in both groups made their decision early in the recruitment process. Considerations of reasons for/against participation differed by enrollment status. CONCLUSIONS: Understanding how parents experience recruitment, and how this differs by enrollment status, may help researchers improve recruitment processes for families and increase enrollment. The parental experience of recruitment varied by enrollment status. These findings can guide future work aiming to inform optimal recruitment strategies for neonatal clinical trials.

Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung.

In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis-infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection.

Lnc(ing)RNAs to the "shock and kill" strategy for HIV-1 cure.

The advent of antiretroviral therapy almost 25 years ago has transformed HIV-1 infection into a manageable chronic condition, albeit still incurable. The inability of the treatment regimen to eliminate latently infected cells that harbor the virus in an epigenetically silent state poses a major hurdle. Current cure approaches are focused on a "shock and kill" strategy that uses latency-reversing agents to chemically reverse the proviral quiescence in latently infected cells, followed by immune-mediated clearance of reactivated cells. To date, hundreds of compounds have been investigated for viral reactivation, yet none has resulted in a functional cure. The insufficiency of these latency-reversing agents (LRAs) alone indicates a critical need for additional, alternate approaches such as genetic manipulation. Long non-coding RNAs (lncRNAs) are an emerging class of regulatory RNAs with functional roles in many cellular processes, including epigenetic modulation. A number of lncRNAs have already been implicated to play important roles in HIV-1 latency and, as such, pharmacological modulation of lncRNAs constitutes a rational alternative approach in HIV-1 cure research. In this review, we discuss the current state of knowledge of the role of lncRNAs in HIV-1 infection and explore the scope for a lncRNA-mediated genetic approach within the shock and kill strategy of HIV-1 cure.

Parental Factors Associated With the Decision to Participate in a Neonatal Clinical Trial.

Importance: It remains poorly understood how parents decide whether to enroll a child in a neonatal clinical trial. This is particularly true for parents from racial or ethnic minority populations. Understanding factors associated with enrollment decisions may improve recruitment processes for families, increase enrollment rates, and decrease disparities in research participation. Objective: To assess differences in parental factors between parents who enrolled their infant and those who declined enrollment for a neonatal randomized clinical trial. Design, Setting, and Participants: This survey study conducted from July 2017 to October 2019 in 12 US level 3 and 4 neonatal intensive care units included parents of infants who enrolled in the High-dose Erythropoietin for Asphyxia and Encephalopathy (HEAL) trial or who were eligible but declined enrollment. Data were analyzed October 2019 through July 2020. Exposure: Parental choice of enrollment in neonatal clinical trial. Main Outcomes and Measures: Percentages and odds ratios (ORs) of parent participation as categorized by demographic characteristics, self-assessment of child's medical condition, study comprehension, and trust in medical researchers. Survey questions were based on the hypothesis that parents who enrolled their infant in HEAL differ from those who declined enrollment across 4 categories: (1) infant characteristics and parental demographic characteristics, (2) perception of infant's illness, (3) study comprehension, and (4) trust in clinicians and researchers. Results: Of a total 387 eligible parents, 269 (69.5%) completed the survey and were included in analysis. This included 183 of 242 (75.6%) of HEAL-enrolled and 86 of 145 (59.3%) of HEAL-declined parents. Parents who enrolled their infant had lower rates of Medicaid participation (74 [41.1%] vs 47 [55.3%]; P = .04) and higher rates of annual income greater than $55 000 (94 [52.8%] vs 30 [37.5%]; P = .03) compared with those who declined. Black parents had lower enrollment rates compared with White parents (OR, 0.35; 95% CI, 0.17-0.73). Parents who reported their infant's medical condition as more serious had higher enrollment rates (OR, 5.7; 95% CI, 2.0-16.3). Parents who enrolled their infant reported higher trust in medical researchers compared with parents who declined (mean [SD] difference, 5.3 [0.3-10.3]). There was no association between study comprehension and enrollment. Conclusions and Relevance: In this study, the following factors were associated with neonatal clinical trial enrollment: demographic characteristics (ie, race/ethnicity, Medicaid status, and reported income), perception of illness, and trust in medical researchers. Future work to confirm these findings and explore the reasons behind them may lead to strategies for better engaging underrepresented groups in neonatal clinical research to reduce enrollment disparities.

Dual RNA-Sequencing of Mycobacterium tuberculosis-Infected Cells from a Murine Infection Model.

Dual RNA-sequencing is a powerful technique to assess both bacterial and host transcriptomes in an unbiased way. We developed a protocol to perform Dual RNA-seq on in vivo-derived macrophage populations infected with Mycobacterium tuberculosis. Here, we provide a practical step-by-step guide to execute the protocol on Mtb-infected cells from a murine infection model. Our protocol can also be easily applied to perform Dual RNA-seq on in vitro-derived cells as well as different Mtb-infected host cell types. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2020).

TZM-gfp cells: a tractable fluorescent tool for analysis of rare and early HIV-1 infection.

Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates.

Nutrition and the Gut Microbiota in 10- to 18-Month-Old Children Living in Urban Slums of Mumbai, India.

In this cross-sectional study, we describe the composition and diversity of the gut microbiota among undernourished children living in urban slums of Mumbai, India, and determine how nutritional status, including anthropometric measurements, dietary intakes from complementary foods, feeding practices, and micronutrient concentrations, is associated with their gut microbiota. We collected rectal swabs from children aged 10 to 18 months living in urban slums of Mumbai participating in a randomized controlled feeding trial and conducted 16S rRNA sequencing to determine the composition of the gut microbiota. Across the study cohort, Proteobacteria dominated the gut microbiota at over 80% relative abundance, with Actinobacteria representation at <4%, suggesting immaturity of the gut. Increased microbial α-diversity was associated with current breastfeeding, greater head circumference, higher fat intake, and lower hemoglobin concentration and weight-for-length Z-score. In redundancy analyses, 47% of the variation in Faith's phylogenetic diversity (Faith's PD) could be accounted for by age and by iron and polyunsaturated fatty acid intakes. Differences in community structure (ß-diversity) of the microbiota were observed among those consuming fats and oils the previous day compared to those not consuming fats and oils the previous day. Our findings suggest that growth, diet, and feeding practices are associated with gut microbiota metrics in undernourished children, whose gut microbiota were comprised mainly of Proteobacteria, a phylum containing many potentially pathogenic taxa.IMPORTANCE The impact of comprehensive nutritional status, defined as growth, nutritional blood biomarkers, dietary intakes, and feeding practices, on the gut microbiome in children living in low-resource settings has remained underreported in microbiome research. Among undernourished children living in urban slums of Mumbai, India, we observed a high relative abundance of Proteobacteria, a phylum including many potentially pathogenic species similar to the composition in preterm infants, suggesting immaturity of the gut, or potentially a high inflammatory burden. We found head circumference, fat and iron intake, and current breastfeeding were positively associated with microbial diversity, while hemoglobin and weight for length were associated with lower diversity. Findings suggest that examining comprehensive nutrition is critical to gain more understanding of how nutrition and the gut microbiota are linked, particularly in vulnerable populations such as children in urban slum settings.