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Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions. Bacteria can become dormant or form spores when starved for nutrients. Here, Gray et al. describe an alternative strategy, or ‘oligotrophic growth state’, showing that non-sporulating Bacillus subtilis cells can survive deep starvation conditions by adopting an almost coccoid shape and extremely low growth rates.

The bactericidal activity of several antibiotics partially relies on the production of reactive oxygen species (ROS), which is generally linked to enhanced respiration and requires the Fenton reaction. Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. Here, we use Bacillus subtilis cells in stationary phase, as a model system of dormant cells, to show that pharmacological induction of membrane depolarization enhances the antibiotics’ bactericidal activity and also leads to ROS production. However, in contrast to previous studies, this results primarily in production of superoxide radicals and does not require the Fenton reaction. Genetic analyzes indicate that Rieske factor QcrA, the iron-sulfur subunit of respiratory complex III, seems to be a primary source of superoxide radicals. Interestingly, the membrane distribution of QcrA changes upon membrane depolarization, suggesting a dissociation of complex III. Thus, our data reveal an alternative mechanism by which antibiotics can cause lethal ROS levels, and may partially explain why membrane-targeting antibiotics are effective in eliminating persisters. The bactericidal activity of several antibiotics partially relies on production of reactive oxygen species (ROS), which requires the Fenton reaction. Here, the authors show that membrane depolarization also leads to ROS production, but this occurs via other mechanisms that do not require the Fenton reaction.

Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.

Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

Background African animal trypanosomosis (AAT) is a major livestock disease in Kenya. Even though, over the years various organizations have collected a vast amount of field data on tsetse and AAT in different parts of the country, recent national-level maps are lacking. To address this gap, a national atlas of tsetse and AAT distribution is being developed by the Kenya Tsetse and Trypanosomosis Eradication Council (KENTTEC) and partners. Methods All data collected by KENTTEC from 2006 to 2019 were systematically assembled, georeferenced and harmonized. A comprehensive data repository and a spatially-explicit database were created. Input data were collected mainly in the context of control activities, and include both baseline surveys (i.e. pre-intervention) and the subsequent monitoring during and after interventions. Surveys were carried out in four regions (i.e. Western, Rift Valley, Central and Coast), and in 21 of the 47 counties in Kenya. Various devices were used for entomological data collection (i.e. biconical, NGU and H traps, and sticky panels), while the buffy-coat technique was the method used to detect AAT. Results Tsetse trapping was carried out in approximately 5000 locations, and flies (> 71,000) were caught in all four investigated regions. Six species of Glossina were detected: G. pallidipes (87% of the catches); G. brevipalpis (8%); G. fuscipes fuscipes (4%); G. longipennis (< 1%); G. austeni (< 1%); and G. swynnertoni (< 1%). A total of 49,785 animals (98% of which cattle) were tested for AAT in approximately 500 locations. Of these, 914 animals were found to be infected. AAT was confirmed in all study regions, in particular caused by Trypanosoma vivax (48% of infections) and T. congolense (42%). Fewer cases of T. brucei were found. Conclusions The development and regular update of a comprehensive national database of tsetse and AAT is crucial to guide decision making for the progressive control of the disease. This first version of the atlas based on KENTTEC data has achieved a remarkable level of geographical coverage, but temporal and spatial gaps still exist. Other stakeholders at the national and international level will contribute to the initiative, thus improving the completeness of the atlas.

Female genital schistosomiasis as a result of chronic infection with (commonly known as bilharzia) continues to be largely ignored by national and global health policy-makers. International attention for large-scale action against the disease focuses on whether it is a risk factor for the transmission of human immunodeficiency virus (HIV). Yet female genital schistosomiasis itself is linked to pain, bleeding and sub- or infertility, leading to social stigma, and is a common issue for women in schistosomiasis-endemic areas in sub-Saharan Africa. The disease should therefore be recognized as another component of a comprehensive health and human rights agenda for women and girls in Africa, alongside HIV and cervical cancer. Each of these three diseases has a targeted and proven preventive intervention: antiretroviral therapy and pre-exposure prophylaxis for HIV; human papilloma virus vaccine for cervical cancer; and praziquantel treatment for female genital schistosomiasis. We discuss how female genital schistosomiasis control can be integrated with HIV and cervical cancer care. Such a programme will be part of a broader framework of sexual and reproductive health and rights, women's empowerment and social justice in Africa. Integrated approaches that join up multiple public health programmes have the potential to expand or create opportunities to reach more girls and women throughout their life course. We outline a pragmatic operational research agenda that has the potential to optimize joint implementation of a package of measures responding to the specific needs of girls and women.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

To date, there is no licensed vaccine against the protozoan parasite Trypanosoma cruzi ( T. cruzi) , the etiological agent of Chagas Disease. T. cruzi has evolved numerous mechanisms to evade and manipulate the host immune system. Among the subversive strategies employed by the parasite, marked increases in CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSCs) in several organs have been described. We have reported that CD11b+ Gr-1+ cells are involved not only during infection but also after immunization with a trans-sialidase fragment (TSf) adjuvanted with a cage-like particle adjuvant (ISPA). Thus, the aim of this work was to gain control over the involvement of MDSCs during immunization to potentiate a vaccine candidate with protective capacity in multiple mouse models of T. cruzi infection. Here, we show that the Gr-1+ cells that increase during TSf-ISPA immunization have suppressive capacity over bone marrow-derived dendritic cells and CD4+ lymphocytes. Protocols using one or two doses of 5-fluorouracil (5FU) were employed to deplete and control MDSC dynamics during immunization. The protocol based on two doses of 5FU (double 5FU TSf-ISPA) was more successful in controlling MDSCs during immunization and triggered a higher immune effector response, as evidenced by increased numbers of CD4+, CD4+CD44+, CD8+, CD8+CD44+, CD11c+, and CD11c+CD8α+ cells in the spleen and lymph nodes of double 5FU TSf-ISPA mice as compared to 5FU-TSf-ISPA mice. In line with these results, the protective capacity of the double 5FU TSf-ISPA protocol was higher compared to the 5FU-TSf-ISPA protocol against high lethal doses of intraperitoneal infection with the Tulahuen T. cruzi strain. When cross-protective capacity was analyzed, the optimized protocol based on double 5FU TSf-ISPA conferred protection in several preclinical models using different discrete typing units (DTU VI and DTU I), different mouse strains (BALB/c and C57BL/6), different parasite doses (1000 to 20000), and routes of administration (intraperitoneal and intradermal). Developing vaccines that are currently lacking may require new strategies to further potentiate vaccine candidates. Results reported herein provide evidence that rational control of cells from the regulatory arm of the immune system could enhance a vaccine candidate with cross-protective capacity in multiple mouse models of T. cruzi infection.

Summary Background Colonoscopy is a screening modality for the early detection of colonic polyps and cancers but is underutilized, particularly among minorities. Objective To identify potential barriers to screening colonoscopy among low income Latino and white non-Latino patients in an urban community health center. Design, participants, and approach We conducted semistructured interviews with a convenience sample of patients 53 to 70 years old, eligible for colorectal cancer screening that spoke English or Spanish. Open-ended questions explored knowledge, beliefs, and experience with or reasons for not having screening colonoscopy. We performed content analysis of transcripts using established qualitative techniques. Results Of 40 participants recruited, 57% were women, 55% Latino, 20% had private health insurance, and 40% had a prior colonoscopy. Participants described a wide range of barriers categorized into 5 major themes: (1) System barriers including scheduling, financial, transportation, and language difficulties; (2) Fear of pain or complications of colonoscopy and fear of diagnosis (cancer); (3) Lack of desire or motivation , including “laziness” and “procrastination”; (4) Dissuasion by others influencing participants’ decision regarding colonoscopy; and (5) Lack of provider recommendation including not hearing about colonoscopy or not understanding the preparation instructions. Conclusions Understanding of the range of barriers to colorectal cancer screening can help develop multimodal interventions to increase colonoscopy rates for all patients including low-income Latinos. Interventions including systems improvements and navigator programs could address barriers by assisting patients with scheduling, insurance issues, and transportation and providing interpretation, education, emotional support, and motivational interviewing.