Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRS_Mabs and LeuRS_Mtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.     

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC₅ prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.     

Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Complex

Purpose

Clarithromycin was considered the cornerstone for the treatment of Mycobacterium abscessus complex infections. Genetic resistance mechanisms have been described and many experts propose amikacin as an alternative. Nevertheless, clarithromycin has several advantages; therefore, it is necessary to identify the non-functional erm(41) allele to determine the most suitable treatment. The aims of this study were to characterize the molecular mechanisms of clarithromycin resistance in a collection of Mycobacterium abscessus complex isolates and to verify the relationship between these mechanisms and the antibiogram.

Materials and Methods

Clinical isolates of M. abscessus complex (n = 22) from 16 patients were identified using four housekeeping genes (rpoB, secA1, sodA and hsp65), and their genetic resistance was characterized by studying erm(41) and rrl genes. Nine strains were recovered from the clinical isolates and subjected to E-test and microdilution clarithromycin susceptibility tests, with readings at 3, 7 and 14 days.

Results

We classified 11/16 (68.8%) M. abscessus subsp. abscessus, 4/16 (25.0%) M. abscessus subsp. bolletii, and 1/16 (6.3%) M. abscessus subsp. massiliense. T28 erm(41) allele was observed in 8 Mycobacterium abscessus subps. abscessus and 3 Mycobacterium abscessus subsp. bolletii. One strain of M. abscessus subsp. bolletii had an erm(41) gene truncated and was susceptible to clarithromycin. No mutations were observed in rrl gene first isolates. In three patients, follow-up of initial rrl wild-type strains showed acquired resistance.

Conclusions

Most clinical isolates of M. abscessus complex had inducible resistance to clarithromycin and total absence of constitutive resistance. Our findings showed that the acquisition of resistance mutations in rrl gene was associated with functional and non-functional erm(41) gene. Caution is needed when using erm(41) sequencing alone to identify M. abscessus subspecies. This study reports an acquired mutation at position 2057 of rrl gene, conferring medium-low clarithromycin constitutive resistance.     

Mycobacterium abscessus Induces a Limited Pattern of Neutrophil Activation That Promotes Pathogen Survival

Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.     

Pathogenic Nontuberculous Mycobacteria Resist and Inactivate Cathelicidin: Implication of a Novel Role for Polar Mycobacterial Lipids

Nontuberculous mycobacteria (NTM) are a large group of environmental organisms with worldwide distribution, but only a relatively few are known to be pathogenic. Chronic, debilitating lung disease is the most common manifestation of NTM infection, which is often refractory to treatment. The incidence and prevalence of NTM lung disease are increasing in the United States and in many parts of the world. Hence, a more complete understanding of NTM pathogenesis will provide the foundation to develop innovative approaches to treat this recalcitrant disease. Herein, we demonstrate that several species of NTM show broad resistance to the antimicrobial peptide, cathelicidin (LL-37). Resistance to LL-37 was not significantly different between M. avium that contain serovar-specific glycopeptidolipid (GPL, M. avium^ssGPL) and M. avium that do not (M. avium^ΔssGPL). Similarly, M. abscessus containing non-specific GPL (M. abscessus^nsGPL(+)) or lacking nsGPL (M. abscessus^nsGPL(-)) remained equally resistant to LL-37. These findings would support the notion that GPL are not the components responsible for NTM resistance to LL-37. Unexpectedly, the growth of M. abscessus^nsGPL(-) increased with LL-37 or scrambled LL-37 peptide in a dose-dependent fashion. We also discovered that LL-37 exposed to NTM had reduced antimicrobial activity, and initial work indicates that this is likely due to inactivation of LL-37 by lipid component(s) of the NTM cell envelope. We conclude that pathogenic NTM resist and inactivate LL-37. The mechanism by which NTM circumvent the antimicrobial activity of LL-37 remains to be determined.     

Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

Background

Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.

Results

Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.

Conclusion

We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1569-2) contains supplementary material, which is available to authorized users.     

Comparative analysis of immune responses to Mycobacterium abscessus infection and its antigens in two murine models

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Because little is known regarding immune responses elicited by M. abscessus or its antigens, immunological responses were studied in two murine models subjected to intravenous (high-dose or systemic infection) or pulmonary (low-dose or local infection) inoculation with M. abscessus ATCC 19977. An overall comparison between the two models showed similar patterns of bacterial survival and host immune responses. The colonization of M. abscessus was the highest at 5 days post-infection (dpi) and its elimination was positively correlated with cell-mediated immunity in both challenges. However, an inverse relationship was observed between progressive inflammation and mycobacterial colonization levels in mice infected with a high dose at 14 dpi. Regarding antigens, culture filtrate (CF) of M. abscessus strongly induced IFN-γ secretion, whereas cellular extract (CE) antigen elicited strong antibody responses. The antibody response to M. abscessus antigens in mice subjected to low-dose infection increased when the cellular immune response decreased over 14 dpi. However, the antibody response for the high-dose infection increased promptly after the infection. In comparison of cytokine expression in lung homogenates after M. abscessus infection, Thl and Th2 cytokines increased simultaneously in the high-dose infection, whereas only cell-mediated immunity developed in the low-dose pulmonary infection. These findings not only enhance our understanding of the immune response to M. abscessus infection according to systemic or pulmonary infection, but may also aid in immunological diagnosis and vaccine development. M. abscessus, murine infection model, immune response, antigens, cytokines     

A β-Lactamase based reporter system for ESX dependent protein translocation in mycobacteria

Protein secretion is essential for all bacteria in order to interact with their environment. Mycobacterium tuberculosis depends on protein secretion to subvert host immune response mechanisms. Both the general secretion system (Sec) and the twin-arginine translocation system (Tat) are functional in mycobacteria. Furthermore, a novel type of protein translocation system named ESX has been identified. In the genome of M. tuberculosis five paralogous ESX regions (ESX-1 to ESX-5) have been found. Several components of the ESX translocation apparatus have been identified over the last ten years. The ESX regions are composed of a basic set of genes for the translocation machinery and the main substrate - a heterodimer. The best studied of these heterodimers is EsxA (ESAT-6)/EsxB (CFP-10), which has been shown to be exported by ESX-1. EsxA/B is heavily involved in virulence of M. tuberculosis. EsxG/H is exported by ESX-3 and seems to be involved in an essential iron-uptake mechanism in M. tuberculosis. These findings make ESX-3 components high profile drug targets. Until now, reporter systems for determination of ESX protein translocation have not been developed. In order to create such a reporter system, a truncated β-lactamase ('bla TEM-1) was fused to the N-terminus of EsxB, EsxG and EsxU, respectively. These constructs have then been tested in a β-lactamase (BlaS) deletion strain of Mycobacterium smegmatis. M. smegmatis ΔblaS is highly susceptible to ampicillin. An ampicillin resistant phenotype was conferred by translocation of Bla TEM-1-Esx fusion proteins into the periplasm. BlaTEM-1-Esx fusion proteins were not found in the culture filtrate suggesting that plasma membrane translocation and outer membrane translocation are two distinct steps in ESX secretion. Thus we have developed a powerful tool to dissect the molecular mechanisms of ESX dependent protein translocation and to screen for novel components of the ESX systems on a large scale.     

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp Polybia dimorpha

Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.     

The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.     

The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.     

Structures of EccB<Subscript>1</Subscript> and EccD<Subscript>1</Subscript> from the core complex of the mycobacterial ESX-1 type VII secretion system

Background

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system.

Results

This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB₁ and EccD₁. The periplasmic domain of EccB₁ consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB₁ are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD₁has a ubiquitin-like fold and forms a dimer with a negatively charged groove.

Conclusions

These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.

     

Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence

The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp⁵⁵ (W55) or Gly⁵⁷ (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe⁵⁰ (F50) and Lys⁶² (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe⁵ (F5) and Lys⁴¹ (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.     

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.     

Mycobacterium abscessus Morphotype Comparison in a Murine Model

Pulmonary infections with Mycobacterium abscessus (M. abscessus) are increasingly prevalent in patients with lung diseases such as cystic fibrosis. M. abscessus exists in two morphotypes, smooth and rough, but the impact of morphotype on virulence is unclear. We developed an immune competent mouse model of pulmonary M. abscessus infection and tested the differences in host inflammatory response between the morphotypes of M. abscessus. Smooth and rough morphotypes of M. abscessus were isolated from the same American Type Culture Collection strain. Wild type and cystic fibrosis mice were intratracheally inoculated with known quantities of M. abscessus suspended in fibrin plugs. At the time of sacrifice lung and splenic tissues and bronchoalveolar lavage fluid were collected and cultured. Bronchoalveolar lavage fluid was analyzed for leukocyte count, differential and cytokine expression. Pulmonary infection with M. abscessus was present at both 3 days and 14 days post-inoculation in all groups at greater levels than systemic infection. Inoculation with M. abscessus rough morphotype resulted in more bronchoalveolar lavage fluid neutrophils compared to smooth morphotype at 14 days post-inoculation in both wild type (p = 0.01) and cystic fibrosis (p<0.01) mice. Spontaneous in vivo conversion from smooth to rough morphotype occurred in 12/57 (21%) of mice. These mice trended towards greater weight loss than mice in which morphotype conversion did not occur. In the described fibrin plug model of M. abscessus infection, pulmonary infection with minimal systemic dissemination is achieved with both smooth and rough morphotypes. In this model M. abscessus rough morphotype causes a greater host inflammatory response than the smooth based on bronchoalveolar lavage fluid neutrophil levels.     

Limited Contribution of IL-36 versus IL-1 and TNF Pathways in Host Response to Mycobacterial Infection

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M. bovis BCG infection and virulent aerogenic M. tuberculosis infection. IL-36γ expression was increased in the lung of M. bovis BCG infected mice. However, IL-36R deficient mice infected with M. bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M. tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection.     

Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis ‘knock-in’ strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.