Biology subjects

Ioerger, T. R.

Publications and source records attributed to Ioerger, T. R..

28 records · Page 2Linked to original sources

bioRxiv2020

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice

The outcome of an encounter with Mycobacterium tuberculosis depends on the pathogens ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that many virulence pathways are only in specific host microenvironments, identifying the large fraction of the pathogens genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, identifying the specific host-pathogen genetic interactions that influence pathogenesis.

genetics↗

bioRxiv2020

Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

microbiology↗

bioRxiv2020

Cell cycle-associated expression patterns predict gene function in mycobacteria

While the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly-growing bacteria, DNA replication and cell division are temporally-resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine if cell-cycle dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the IMP dehydrogenase guaB2 dedicated to guanosine synthesis displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division, and identify periodically regulated genes that can be related to specific cell biological functions.

microbiology↗

bioRxiv2019

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control requires a sequence motif in the 5 region of the porin transcript. Thus, LepA controls the abundance of specific proteins, likely through its activity during translation. ImportanceOur understanding of how ribosomes properly synthesis an entire cellular proteome, in all its complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the use of GTPases such as LepA, that have evolved to aid in specific protein synthesis.

microbiology↗

bioRxiv2019

Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a marginal potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

microbiology↗

bioRxiv2019

Statistical Analysis of Variability in TnSeq Data Across Conditions Using Zero-Inflated Negative Binomial Regression

Deep sequencing of transposon mutant libraries (or TnSeq) is a powerful method for probing essentiality of genomic loci under different environmental conditions. Various analytical methods have been described for identifying conditionally essential genes whose tolerance for insertions varies between two conditions. However, for large-scale experiments involving many conditions, a method is needed for identifying genes that exhibit significant variability in insertions across multiple conditions. In this paper, we introduce a novel statistical method for identifying genes with significant variability of insertion counts across multiple conditions based on Zero-Inflated Negative Binomial (ZINB) regression. Using likelihood ratio tests, we show that the ZINB fits TnSeq data better than either ANOVA or a Negative Binomial (in a generalized linear model). We use ZINB regression to identify genes required for infection of M. tuberculosis H37Rv in C57BL/6 mice. We also use ZINB to perform a retrospective analysis of genes conditionally essential in H37Rv cultures exposed to multiple antibiotics. Our results show that, not only does ZINB generally identify most of the genes found by pairwise resampling (and vastly out-performs ANOVA), but it also identifies additional genes where variability is detectable only when the magnitudes of insertion counts are treated separately from local differences in saturation, as in the ZINB model.

bioinformatics↗

bioRxiv2019

Global assessment of Mycobacterium avium subspecies hominissuis genetic requirement for growth and virulence.

Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth, and organized ~3500 identified transposon mutants for hypothesis-driven research. The majority (71 %) of the genes we identified as essential for MAH in vitro had a growth-essential mutual ortholog in the related and highly virulent M. tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. Strikingly, > 97 % of the MAH virulence genes had a mutual ortholog in Mtb. Two of the three virulence genes specific to MAH (i.e. no Mtb mutual orthologs) were PPE proteins, a family of proteins unique to mycobacteria and highly associated with virulence. Finally, we validated novel genes as required for successful MAH infection; one encoding a probable MFS transporter and another a hypothetical protein located in immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.\n\nAuthor summaryPulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the M. avium complex (MAC), whereof >90% arise from Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention and diagnostics, we need to understand the underlying mechanisms of infection. While genetic manipulation of pathogens is crucial to study pathogenesis, M. avium (Mav) has been found notoriously hard to engineer. Here, we identify an MAH strain highly susceptible to high-density transposon mutagenesis and transformation, facilitating genetic engineering and analysis of gene function. We provide crucial insights into this strains global genetic requirements for growth and infection. Surprisingly, we find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment, or translate to broader treatment of related mycobacterial diseases.

microbiology↗

bioRxiv2018

New inhibitors of Mycobacterium tuberculosis identified using systems chemical biology

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECTs ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

microbiology↗

bioRxiv2018

Maturing Mycobacterial Peptidoglycan Required Non-canonical Crosslinks to Maintain Shape

In most well studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin binding proteins (PBPs). However, in mycobacteria, L,D-transpeptidase (LDT)-mediated crosslinks are highly abundant. To elucidate the role of these unusual crosslinks, we characterized mycobacterial cells lacking all LDTs. We find that LDT-mediated crosslinks are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT-inhibition. Thus, knowledge about the single-cell distribution of drug targets can be exploited to more effectively treat this pathogen.

microbiology↗

bioRxiv2018

Identification of cyclic hexapeptides natural products with inhibitory potency against Mycobacterium tuberculosis

A set of [~]500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3-8.9 M. Eleven structural analogs had no significant activity (MIC > 20 M) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.

microbiology↗