Magnus Steigedal

Members of theMycobacterium aviumcomplex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome.

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. Author summary: Pulmonary 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 strain’s 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.

Mycobacterium avium (Mav) complex (MAC) are characterized as non-tuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact on the infection outcome. The current study aimed to characterize the genomic changes within Mav isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed field gel electrophoresis and whole genome sequencing was performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Patients (4, 9 and 13) who contributed more than two isolates were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single nucleotide polymorphisms (SNPs), suggesting that Mav accumulates mutations at high rates during persistent infections. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency towards increased persistence and down-regulation of inflammatory cytokines by host-adapted Mav strains. The study revealed rapid genetic evolution of Mav in chronically infected patients accompanied with change in virulence properties of the sequential mycobacterial isolates. IMPORTANCE: MAC are a group of opportunistic pathogens, consisting of Mav and M. intracellulare species. Mav is found ubiquitously in the environment. In Mav infected individuals, Mav has been known to persist for long periods of time, and anti-mycobacterial drugs are unable to effectively clear the infection. The continued presence of the bacteria, could be attributed to either a single persistent strain or reinfection with the same or different strain. We examined sequential isolates collected over time from Mav infected individuals and observed that most patients carried the same strain overtime and were not re infected. We observed high rates of mutation within the serial isolates, accompanied with changes in virulence properties. In the light of increase in incidence of MAC related infections, this study highlights the possibility that host adapted Mav undergo genetic modifications to cope with the host environment and thereby persisting longer.

Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.

Significance: Inflammatory signaling is a central mechanism controlling host defenses to pathogens. Members of Mycobacterium avium complex cause disease in immunocompromised patients and in individuals with predisposing lung abnormalities. We provide evidence of a mechanism in human primary macrophages whereby the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) negatively regulates inflammatory responses and thus facilitates intracellular growth of M. avium . Our findings are of high biological and clinical significance, as opportunistic infections with nontuberculous mycobacteria are receiving renewed attention because of increased incidence and difficulties in treatment. Altered Keap1 gene expression may also have vital clinical implications for other inflammation-associated conditions, opening novel research venues for translational research, for instance in the expanding field of host-targeted therapy for infectious diseases.

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.

Competition for iron is a critical component of successful bacterial infections, but the underlying in vivo mechanisms are poorly understood. We have previously demonstrated that lipocalin 2 (LCN2) is an innate immunity protein that binds to bacterial siderophores and starves them for iron, thus representing a novel host defense mechanism to infection. In the present study we show that LCN2 is secreted by the urinary tract mucosa and protects against urinary tract infection (UTI). We found that LCN2 was expressed in the bladder, ureters, and kidneys of mice subject to UTI. LCN2 was protective with higher bacterial numbers retrieved from bladders of Lcn2-deficient mice than from wild-type mice infected with the LCN2-sensitive Escherichia coli strain H9049. Uropathogenic E. coli mutants in siderophore receptors for salmochelin, aerobactin, or yersiniabactin displayed reduced fitness in wild-type mice, but not in mice deficient of LCN2, demonstrating that LCN2 imparts a selective pressure on bacterial growth in the bladder. In a human cohort of women with recurrent E. coli UTIs, urine LCN2 levels were associated with UTI episodes and with levels of bacteriuria. The number of siderophore systems was associated with increasing bacteriuria during cystitis. Our data demonstrate that LCN2 is secreted by the urinary tract mucosa in response to uropathogenic E. coli challenge and acts in innate immune defenses as a colonization barrier that pathogens must overcome to establish infection.

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 MycP ₃ of M. smegmatis , is partially expendable. All of the esx-3 mutants tested, including the Δ mycP _3ms mutant, failed to export the native Esx-3 substrates EsxH _ms and EsxG _ms 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 EsxH _ms and EsxG _ms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxG _ms and EsxH _ms secretion in the ΔmycP _3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro . We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

Opportunistic infections with non-tuberculous mycobacteria such as Mycobacterium avium are receiving renewed attention because of increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanisms contributing to containment in a mouse infection model using virulent M. avium strain 104 in C57BL/6 mice. M. avium caused chronic infection in mice, as shown by sustained organ bacterial load. In the liver, bacteria were contained in granuloma-like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in the adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti-mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon-γ were found in infected liver, spleen and serum of mice approximately 2 weeks post infection and coincided with a halt in organ bacterial growth. In contrast, expression of tumour necrosis factor was surprisingly low in spleen compared with liver. We did not detect interleukin-17 in infected organs or M. avium-specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens. This detailed characterization of M. avium infection in C57BL/6 mice may provide a basis for future studies aimed at gaining better insight into mechanisms leading to containment of infections with non-tuberculous mycobacteria.

Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein