Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems

A Novel Antimycobacterial Compound Acts as an Intracellular Iron Chelator

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound

BKV Agnoprotein Interacts with α-Soluble N-Ethylmaleimide-Sensitive Fusion Attachment Protein, and Negatively Influences Transport of VSVG-EGFP

Background: The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. Principal Findings: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them a-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and a-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and a-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with a-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. Conclusions: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with a-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter

Global Assessment of Mycobacterium avium subsp. hominissuis Genetic Requirement for Growth and Virulence

publishedVersio

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner

Global Assessment of Mycobacterium avium subsp. 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 ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb. Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the 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

Regulation of the zeocin resistance gene in the presence of zeocin.

<p>(A) <i>Msmeg</i> transformed with pMDX-zeo was grown to OD₆₀₀ 0.05–0.1 before addition of 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) was added. Cells were then incubated for 5 hours at 30°C. The cells were normalized by OD₆₀₀, serially diluted, and spotted on plates containing increasing amounts of zeocin and 1.5 mM <i>m</i>-toluate (induced) or ethanol (uninduced) and incubated at 30°C for 2 days. (B) <i>Msmeg</i> transformed with pMDX-zeo was pre-induced for 5 hours as described above, then diluted to OD₆₀₀ 0.005 and grown in triplicates in micro-plate wells in the presence of increasing concentrations of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200 and 250 μg/ml) and 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) shaking at 37°C. Growth was monitored by Bioscreen, registering OD₆₀₀ every other hour. The samples are presented by the OD₆₀₀ of uninduced or induced <i>Msmeg</i> pMDX-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase. Error bars represent standard deviations and the results represent three independent experiments.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Benzoic acid-inducible expression system, XylS/<i>Pm</i>, for regulation of genes in mycobacteria.

<p>The inducible <i>Pm</i> promoter and its activator XylS regulate the expression of “your favorite gene” (YFG). XylS is constitutively expressed under control of <i>Ptet</i> in the absence of anhydro-tetracycline (atc) and binds the <i>Pm</i> promoter in the presence of the inducer <i>m</i>-toluate. This facilitates expression of YFG and leaves the expression system ON (upper panel). In the absence of <i>m</i>-toluate, XylS is not activated, leaving the expression system OFF, as expression from <i>Pm</i> is not induced (middle panel). Reverse TetR is constitutively expressed by <i>Psmyc</i>, and binds the operator in <i>Ptet</i> in the presence of atc blocking transcription of <i>xylS</i>. Addition of atc leaves the system in a more fully OFF mode as potential basal <i>Pm</i>-mediated transcription caused by excessive levels of XylS is abolished (lower panel).</p