The use of single-cell RNA-seq to study heterogeneity at varying levels of virus-host interactions.

The outcome of viral infection depends on the diversity of the infecting viral population and the heterogeneity of the cell population that is infected. Until almost a decade ago, the study of these dynamic processes during viral infection was challenging and limited to certain targeted measurements. Presently, with the use of single-cell sequencing technology, the complex interface defined by the interactions of cells with infecting virus can now be studied across the breadth of the transcriptome in thousands of individual cells simultaneously. In this review, we will describe the use of single-cell RNA sequencing (scRNA-seq) to study the heterogeneity of viral infections, ranging from individual virions to the immune response between infected individuals. In addition, we highlight certain key experimental limitations and methodological decisions that are critical to analyzing scRNA-seq data at each scale.

Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export.

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection.

Growth and division mechanisms by which genetic resisters emerge from the rifampicin-surviving population of differentially antibiotic-susceptible mycobacterial subpopulations.

Background: We recently reported the de novo emergence of unusually high numbers of antibiotic resisters from the in vitro cultures of Mycobacterium tuberculosis and Mycobacterium smegmatis surviving in the presence of minimum bactericidal concentration (MBC) of antituberculosis antibiotics. The resisters emerged due to multiple asymmetric divisions of elongated mother cells containing multiple nucleoids and multiple septae. We had earlier found a minor subpopulation of short-sized cells (SCs) and a major subpopulation of normal-sized cells (NCs) (10% and 90%, respectively, of the whole population), with significant difference in antibiotic susceptibility and resister generation frequency, in the in vitro cultures of M. tuberculosis, M. smegmatis, and Mycobacterium xenopi, as well as in pulmonary tuberculosis patients' sputum. However, the mechanisms of growth and division promoting the emergence of antibiotic resisters from these subpopulations remained unknown. Therefore, here, we took up the first-time study to find out the mechanism of growth and division by which antibiotic resisters emerge from the antibiotic-surviving population of the two subpopulations of M. smegmatis. Methods: M. smegmatis SCs and NCs were fractionated from mid-log phase cultures using Percoll gradient centrifugation; their purity was checked and exposed to 10×, 2×, and 0.4× MBC of rifampicin for 120 h. The colony-forming units (CFUs) were determined on rifampicin-free plates for the total population and on rifampicin-containing plates for scoring rifampicin resisters. The phenotype and the morphology of the cells at various stages of the exposure were determined using transmission electron microscopy. The dynamic growth and division mechanisms of the cells to emerge as rifampicin resisters were monitored using live-cell time-lapse imaging. The rifampicin resisters were sequenced for mutations in the rifampicin resistance determining region of rpoB gene. Statistical significance was calculated using two-tailed paired t-test, with *P ≤ 0.05 and **P ≤ 0.01. Results: Multinucleated and multiseptated elongated cells emerged from their respective antibiotic-surviving populations. They produced a large number of sibling-daughter cells through multiple asymmetric divisions in short durations, showing abnormally high spurts in CFUs of antibiotic resisters. The CFUs were several-fold higher than that expected from the mass-doubling time of the subpopulations. Despite this commonality, the subpopulations showed specific differences in their response to different multiples of their respective MBC of rifampicin. Conclusions: Mycobacterial subpopulations come out of rifampicin stress by undergoing multiple nucleoid replications, multiple septation for nucleoid segregation, and acquisition of antibiotic target-specific mutations, followed by multiple asymmetric divisions to generate unusually a large number of rifampicin resisters. Because we had earlier shown that SCs and NCs are present in the pulmonary tuberculosis patients' sputum, the present findings have clinical relevance on the mechanism of emergence of antibiotic-resistant strains from mycobacterial subpopulations.

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation.

The physiological role of mono-ADP-ribosyl transferase (Arr) of Mycobacterium smegmatis, which inactivates rifampicin, remains unclear. An earlier study reported increased expression of arr during oxidative stress and DNA damage. This suggested a role for Arr in the oxidative status of the cell and its associated effect on DNA damage. Since reactive oxygen species (ROS) influence oxidative status, we investigated whether Arr affected ROS levels in M. smegmatis. Significantly elevated levels of superoxide and hydroxyl radical were found in the mid-log phase (MLP) cultures of the arr knockout strain (arr-KO) as compared those in the wild-type strain (WT). Complementation of arr-KO with expression from genomically integrated arr under its native promoter restored the levels of ROS equivalent to that in WT. Due to the inherently high ROS levels in the actively growing arr-KO, rifampicin resisters with rpoB mutations could be selected at 0 hr of exposure itself against rifampicin, unlike in the WT where the resisters emerged at 12th hr of rifampicin exposure. Microarray analysis of the actively growing cultures of arr-KO revealed significantly high levels of expression of genes from succinate dehydrogenase I and NADH dehydrogenase I operons, which would have contributed to the increased superoxide levels. In parallel, expression of specific DNA repair genes was significantly decreased, favouring retention of the mutations inflicted by the ROS. Expression of several metabolic pathway genes also was significantly altered. These observations revealed that Arr was required for maintaining a gene expression profile that would provide optimum levels of ROS and DNA repair system in the actively growing M. smegmatis.

A method for the enrichment, isolation and validation of Mycobacterium smegmatis population surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin.

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

Unique Mode of Cell Division by the Mycobacterial Genetic Resister Clones Emerging De Novo from the Antibiotic-Surviving Population.

The emergence of antibiotic genetic resisters of pathogenic bacteria poses a major public health challenge. The mechanism by which bacterial antibiotic genetic resister clones formed de novo multiply and establish a resister population remained unknown. Here, we delineated the unique mode of cell division of the antibiotic genetic resisters of Mycobacterium smegmatis and Mycobacterium tuberculosis formed de novo from the population surviving in the presence of bactericidal concentrations of rifampicin or moxifloxacin. The cells in the rifampicin/moxifloxacin-surviving population generated elevated levels of hydroxyl radical-inflicting mutations. The genetic mutants selected against rifampicin/moxifloxacin became multinucleated and multiseptated and developed multiple constrictions. These cells stochastically divided multiple times, producing sister-daughter cells phenomenally higher in number than what could be expected from their generation time. This caused an abrupt, unexpectedly high increase in the rifampicin/moxifloxacin resister colonies. This unique cell division behavior was not shown by the rifampicin resisters formed naturally in the actively growing cultures. We could detect such abrupt increases in the antibiotic resisters in others' and our earlier data on the antibiotic-exposed laboratory/clinical M. tuberculosis strains, M. smegmatis and other bacteria in in vitro cultures, infected macrophages/animals, and tuberculosis patients. However, it went unnoticed/unreported in all those studies. This phenomenon occurring in diverse bacteria surviving against different antibiotics revealed the broad significance of the present study. We speculate that the antibiotic-resistant bacillary clones, which emerge in patients with diverse bacterial infections, might be using the same mechanism to establish an antibiotic resister population quickly in the continued presence of antibiotics.IMPORTANCE The bacterial pathogens that are tolerant to antibiotics and survive in the continued presence of antibiotics have the chance to acquire genetically resistant mutations against the antibiotics and emerge de novo as antibiotic resisters. Once the antibiotic resister clone has emerged, often with compromise on growth characteristics, for the protection of the species, it is important to establish an antibiotic-resistant population quickly in the continued presence of the antibiotic. In this regard, the present study has unraveled multinucleation and multiseptation followed by multiple constrictions as the cellular processes used by the bacteria for quick multiplication to establish antibiotic-resistant populations. The study also points out the same phenomenon occurring in other bacterial systems investigated in our laboratory and others' laboratories. Identification of these specific cellular events involved in quick multiplication offers additional cellular processes that can be targeted in combination with the existing antibiotics' targets to preempt the emergence of antibiotic-resistant bacterial strains.

Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic.

Majority of the cells in the bacterial populations exposed to lethal concentrations of antibiotics for prolonged duration succumbs to the antibiotics' sterilizing activity. The remaining cells survive by diverse mechanisms that include reduced permeability of the antibiotics. However, in the cells surviving in the continued presence of lethal concentrations of antibiotics, it is not known whether any cell surface alterations occur that in turn may reduce permeability of the antibiotics. Here we report the presence of a highly negatively charged, hydrophilic, thickened capsular outer layer (TCOL) on a small proportion of the rifampicin surviving population (RSP) of Mycobacterium tuberculosis (Mtb) cells upon prolonged continuous exposure to bactericidal concentrations of rifampicin in vitro. The TCOL reduced the intracellular entry of 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), a fluorochrome-conjugated rifampicin permeability probe of negligible bacteriocidal activity but comparable properties. Gentle mechanical removal of the TCOL enabled significant increase in the 5-FAM-rifampicin permeability. Zeta potential measurements of the cells' surface charge and hexadecane assay for cell surface hydrophobicity showed that the TCOL imparted high negative charge and polar nature to the cells' surface. Flow cytometry using the MLP and RSP cells, stained with calcofluor white, which specifically binds glucose/mannose units in ß (1 → 4) or ß (1 → 3) linkages, revealed the presence of lower content of polysaccharides containing such residues in the TCOL. GC-MS analyses of the TCOL and the normal capsular outer layer (NCOL) of MLP cells showed elevated levels of α-D-glucopyranoside, mannose, arabinose, galactose, and their derivatives in the TCOL, indicating the presence of high content of polysaccharides with these residues. We hypothesize that the significantly high thickness and the elevated negative charge of the TCOL might have functioned as a physical barrier restricting the permeability of the relatively non-polar rifampicin. This might have reduced intracellular rifampicin concentration enabling the cells' survival in the continued presence of high doses of rifampicin. In the context of our earlier report on the de novo emergence of rifampicin-resistant genetic mutants of Mtb from the population surviving under lethal doses of the antibiotic, the present findings attain clinical significance if a subpopulation of the tubercle bacilli in tuberculosis patients possesses TCOL.

Mycobacterium smegmatis moxifloxacin persister cells produce high levels of hydroxyl radical, generating genetic resisters selectable not only with moxifloxacin, but also with ethambutol and isoniazid.

Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing Mycobacterium smegmatis as a model organism for Mycobacterium tuberculosis biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of M. tuberculosis. Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log10 higher frequency from the persister population. Luria-Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged de novo from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant M. tuberculosis. Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.

Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility.

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute ~10-20 % of the population, and the high-buoyant-density normal/long-sized cells (NCs), which form ~80-90 % of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H2O2 and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H2O2-exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria.

Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress.

The present study shows the existence of two specific sub-populations of Mycobacterium smegmatis and Mycobacterium tuberculosis cells differing in size and density, in the mid-log phase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained short-sized cells (SCs) generated through highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells and symmetric cell divisions (SCD) of short-sized mother cells. The other sub-population (~90% of the total population) contained normal/long-sized cells (NCs). The SCs were acid-fast stainable and heat-susceptible, and contained high density of membrane vesicles (MVs, known to be lipid-rich) on their surface, while the NCs possessed negligible density of MVs on the surface, as revealed by scanning and transmission electron microscopy. Percoll density gradient fractionation of MLP cultures showed the SCs-enriched fraction (SCF) at lower density (probably indicating lipid-richness) and the NCs-enriched fraction (NCF) at higher density of percoll fractions. While live cell imaging showed that the SCs and the NCs could grow and divide to form colony on agarose pads, the SCF, and NCF cells could independently regenerate MLP populations in liquid and solid media, indicating their full genomic content and population regeneration potential. CFU based assays showed the SCF cells to be significantly more susceptible than NCF cells to a range of concentrations of rifampicin and isoniazid (antibiotic stress), H2O2 (oxidative stress),and acidified NaNO2 (nitrite stress). Live cell imaging showed significantly higher susceptibility of the SCs of SC-NC sister daughter cell pairs, formed from highly-deviated ACD of normal/long-sized mother cells, to rifampicin and H2O2, as compared to the sister daughter NCs, irrespective of their comparable growth rates. The SC-SC sister daughter cell pairs, formed from the SCDs of short-sized mother cells and having comparable growth rates, always showed comparable stress-susceptibility. These observations and the presence of M. tuberculosis SCs and NCs in pulmonary tuberculosis patients' sputum earlier reported by us imply a physiological role for the SCs and the NCs under the stress conditions. The plausible reasons for the higher stress susceptibility of SCs and lower stress susceptibility of NCs are discussed.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro.

Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.