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

The Azotobacter vinelandii mannuronan C5-epimerases: their biological functions and new tools useful for their future in vivo biotechnological application

Alginate, an industrially widely used polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G), is produced by brown algae and certain bacteria (e.g. pseudomonads and Azotobacter vinelandii). The alginate monomers are grouped in blocks of M, G or MG. G-blocks enable the polymer to form gels by crosslinking (using cations like Ca2+). Commercially, alginates are utilized for their viscosifying and gel-forming properties. At present all commercially available alginates are harvested from brown algae, but with an ever increasing range of possible applications for the polymer alginate production from bacteria are now also being investigated. Alginate in A. vinelandii is produced as an exopolysaccharide released into the growth medium in vegetatively growing cells, but under certain adverse environmental conditions the organism is able to undergo a differentiation process by which it develops into a desiccation resistant and morphologically distinct form designated cyst, which is surrounded by a rigid coat in which alginate is a major component. The alginate from both vegetatively growing cells and cysts contain G-blocks and therefore probably has a potential for commercial production. For all alginate producers the G residues are introduced at the polymer level by mannuronan C5-epimerases. A. vinelandii encode one Ca2+-independent periplasmic epimerase (AlgG) and seven secreted Ca2+-dependent epimerases (AlgE1-7). All the AlgE epimerases have been expressed recombinantly in Escherichia coli and their properties have been extensively studied in vitro. In contrast, much less is known about their in vivo functions and this Ph.D project was undertaken to develop a better understanding of their biological function and to generate a knowledge basis that later may be used to modify A. vinelandii genetically so that it can become an in vivo producer of bacterial alginates with predetermined properties. To elucidate the in vivo roles of the AlgE epimerases in A. vinelandii the genes encoding each of them were individually disrupted. These interruptions had no clear effect on the structure of the alginates produced, or on the morphology of cells grown in shake-flasks (RA1 medium), but when the same cells were grown in fermentors (PM1 medium), the algE3 mutant (strain MS159) alginates contained only 8% G, in contrast to the 25 % found in alginates from wild-type cells grown under the same conditions. This result indicated that the phenotypes of the mutants may be significantly dependent on the growth conditions (Paper 2). Based on the results from the single algE disruptions it appeared likely that more clear effects on the alginate structure and A. vinelandii biology would become more apparent if more than one epimerase gene was inactivated in one single strain. All algE genes with the exception of algE5 are clustered in the A. vinelandii chromosome, and this made it possible to delete the entire cluster, generating strain MS163. In this strain the algE5 gene was then interrupted, generating strain MS163171. Fermentor-grown cells of strain MS163 were found to produce alginates containing only 6% G, and the polymer products from strain MS163171 contained nearly undetectable levels of G (below 2%). In addition strain MS163171 was incapable of forming cystlike structures in RA1 medium and was unable to withstand desiccation in standard cyst-testing experiments. This observation is almost certainly linked to the inability of this strain to make G-blocks (Paper 2). In conclusion, the experiments with the algE mutants for the first time directly showed that the AlgE epimerases are responsible for nearly all alginate epimerization in A. vinelandii. In addition this work shows that the AlgE epimerases are essential for the ability of the cells to differentiate into desiccation resistant cysts (Paper 2). The conclusions reached in these experiments are in complete agreement with the results obtained in a parallel study of a transport system needed for export of the AlgE epimerases. The studies of this system constitute a minor part of this PhD work, but it is important for the independent evidence further substantiating the conclusions on the role of the AlgE epimerases (Paper 1). A very important spin-off of these results is that strain MS163171 seems to represent a very good starting point for production of tailor-made alginates, by constructing a new series of strains in which separate selected algE genes are expressed at carefully controlled levels. Even from the start of this Ph.D project it was realized that to utilize A. vinelandii for production of alginates with predetermined structures, a new gene expression system was needed. No such specialized tools existed then, and it was therefore decided to try to identify and develop a new broad-host-range gene expression system that could be used in A. vinelandii. The new system was found in Acinetobacter sp. and is the promoter of chnB together with its positive regulator ChnR. The system was called Pb. Pb was tested for ability to induce luciferace in response to an externally added inducer and was shown to be inducible in several species (Paper 3). The Pb-promoter was planned to be used in combination with another system previously developed in our group, and which is based on the Pm promoter and its cognate positive regulator XylS. An example of such a combined use is illustrated in paper 3, where the technically more convenient alginate-producer Pseudomonas fluorescens is used as a test organism. This model experiment showed that the two systems in combination could be used to control alginate monomer structure. The prospects for future use of these systems to construct sophisticated production systems for alginate production in A. vinelandii therefore now seem promising

Mycobacterium smegmatis Vaccine Vector Elicits CD4+ Th17 and CD8+ Tc17 T Cells With Therapeutic Potential to Infections With Mycobacterium avium

Mycobacterium avium (Mav) complex is increasingly reported to cause non-tuberculous infections in individuals with a compromised immune system. Treatment is complicated and no vaccines are available. Previous studies have shown some potential of using genetically modified Mycobacterium smegmatis (Msm) as a vaccine vector to tuberculosis since it is non-pathogenic and thus would be tolerated by immunocompromised individuals. In this study, we used a mutant strain of Msm disrupted in EspG3, a component of the ESX-3 secretion system. Infection of macrophages and dendritic cells with Msm ΔespG3 showed increased antigen presentation compared to cells infected with wild-type Msm. Vaccination of mice with Msm ΔespG3, expressing the Mav antigen MPT64, provided equal protection against Mav infection as the tuberculosis vaccine, Mycobacterium bovis BCG. However, upon challenge with Mav, we observed a high frequency of IL-17-producing CD4+ (Th17 cells) and CD8+ (Tc17 cells) T cells in mice vaccinated with Msm ΔespG3::mpt64 that was not seen in BCG-vaccinated mice. Adoptive transfer of cells from Msm ΔespG3-vaccinated mice showed that cells from the T cell compartment contributed to protection from Mav infection. Further experiments revealed Tc17-enriched T cells did not provide prophylactic protection against subsequent Mav infection, but a therapeutic effect was observed when Tc17-enriched cells were transferred to mice already infected with Mav. These initial findings are important, as they suggest a previously unknown role of Tc17 cells in mycobacterial infections. Taken together, Msm ΔespG3 shows promise as a vaccine vector against Mav and possibly other (myco)bacterial infections

Identification and Characterization of an Azotobacter vinelandii Type I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

Alginate is a linear copolymer of β-d-mannuronic acid and its C-5-epimer, α-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (≤2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place

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

publishedVersio

Dynamics of immune effector mechanisms during infection with Mycobacterium aviumin C57BL/6 mice

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 antimycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon-c 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

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