Methylene blue photodynamic therapy of bacterial species found in human abscesses: Planktonic, biofilm, and 3D silicone models.

Deep tissue abscesses are inflammatory, purulent lesions encased in a fibrin-rich pseudocapsule that include multiple bacterial and fungal species. We have initiated a Phase 1 clinical trial exploring the safety and feasibility of methylene blue photodynamic therapy (MB-PDT) at the time of abscess drainage. To optimize treatment parameters for future clinical applications, our goal is to generate physically accurate three-dimensional (3D) abscess models upon which bacteria can be grown. Here, we report results of MB-PDT against four representative bacterial species found in human abscesses in planktonic culture, as biofilms on silicone, and pilot results in 3D silicone molds derived from human abscess computed tomography (CT) images. In all cases, MB-PDT was performed with 665 nm light at a fluence rate of 4 mW/cm2 for 30 minutes, resulting in a fluence of 7.2 J/cm2. In planktonic cultures, MB-PDT was effective against Escherichia coli, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus (MRSA) (4- to 7-fold log CFU reduction). For Klebsiella pneumoniae, increased fluence was required to achieve comparable efficacy. When bacteria were grown as biofilms on silicone, MB-PDT efficacy was reduced (1- to 2-fold CFU reduction). A 3D silicone model was generated based on pelvic abscess CT images, and MRSA was grown in this model for six days. Crystal violet staining showed abundant growth on the silicone, without penetration into the model. These results motivate exploration of both light and drug dose ranging for biofilm samples. Future experiments will additionally focus on MB-PDT of bacteria grown on 3D silicone surfaces.

LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most ß-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

Amphibians as a model to study the role of immune cell heterogeneity in host and mycobacterial interactions.

Mycobacterial infections represent major concerns for aquatic and terrestrial vertebrates including humans. Although our current knowledge is mostly restricted to Mycobacterium tuberculosis and mammalian host interactions, increasing evidence suggests common features in endo- and ectothermic animals infected with non-tuberculous mycobacteria (NTMs) like those described for M. tuberculosis. Importantly, most of the pathogenic and non-pathogenic NTMs detected in amphibians from wild, farmed, and research facilities represent, in addition to the potential economic loss, a rising concern for human health. Upon mycobacterial infection in mammals, the protective immune responses involving the innate and adaptive immune systems are highly complex and therefore not fully understood. This complexity results from the versatility and resilience of mycobacteria to hostile conditions as well as from the immune cell heterogeneity arising from the distinct developmental origins according with the concept of layered immunity. Similar to the differing responses of neonates versus adults during tuberculosis development, the pathogenesis and inflammatory responses are stage-specific in Xenopus laevis during infection by the NTM M. marinum. That is, both in human fetal and neonatal development and in tadpole development, responses are characterized by hypo-responsiveness and a lower capacity to contain mycobacterial infections. Similar to a mammalian fetus and neonates, T cells and myeloid cells in Xenopus tadpoles and axolotls are different from the adult immune cells. Fetal and amphibian larval T cells, which are characterized by a lower T cell receptor (TCR) repertoire diversity, are biased toward regulatory function, and they have distinct progenitor origins from those of the adult immune cells. Some early developing T cells and likely macrophage subpopulations are conserved in adult anurans and mammals, and therefore, they likely play an important role in the host-pathogen interactions from early stages of development to adulthood. Thus, we propose the use of developing amphibians, which have the advantage of being free-living early in their development, as an alternative and complementary model to study the role of immune cell heterogeneity in host-mycobacteria interactions.

Developing Tadpole Xenopus laevis as a Comparative Animal Model to Study Mycobacterium abscessus Pathogenicity.

Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking.

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

Distinct MHC class I-like interacting invariant T cell lineage at the forefront of mycobacterial immunity uncovered in Xenopus.

The amphibian Xenopus laevis is to date the only species outside of mammals where a MHC class I-like (MHC-like) restricted innate-like (i) T cell subset (iVα6 T cells) reminiscent of CD1d-restricted iNKT cells has been identified and functionally characterized. This provides an attractive in vivo model to study the biological analogies and differences between mammalian iT cells and the evolutionarily antecedent Xenopus iT cell defense system. Here, we report the identification of a unique iT cell subset (Vα45-Jα1.14) requiring a distinct MHC-like molecule (mhc1b4.L or XNC4) for its development and function. We used two complementary reverse genetic approaches: RNA interference by transgenesis to impair expression of either XNC4 or the Vα45-Jα1.14 rearrangement, and CRISPR/Cas9-mediated disruption of the Jα1.14 gene segment. Both XNC4 deficiency that ablates iVα45T cell development and the direct disruption of the iVα45-Jα1.14 T cell receptor dramatically impairs tadpole resistance to Mycobacterium marinum (Mm) infection. The higher mortality of Mm-infected tadpoles deficient for iVα45T cells correlates with dysregulated expression responses of several immune genes. In contrast, iVα45-Jα1.14-deficient tadpoles remain fully competent against infection by the ranavirus FV3, which indicates a specialization of this unique iT cell subset toward mycobacterial rather than viral pathogens that involve iVα6 T cells. These data suggest that amphibians, which are evolutionarily separated from mammals by more than 350 My, have independently diversified a prominent and convergent immune surveillance system based on MHC-like interacting innate-like T cells.

galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus.

Mycobacterium abscessus is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the mmpL4b transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in M. abscessus ATCC19977, using a suicide vector bearing the E. coli galK gene and 2-deoxygalactose counterselection. We describe here two variant galK suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the mmpL4b GPL transporter gene, the mbtH GPL biosynthesis gene, the known ß-lactamase gene MAB_2875 and a putative ß-lactamase gene, MAB_2833. We also show that a novel allele of the E. coli aacC4 gene, conferring apramycin resistance (aacC41), can be used as a selectable marker in M. abscessus ATCC19977 at single copy.

The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis.

UNLABELLED: Mycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and are DL-endopeptidases, and RipA has been considered essential to Mycobacterium smegmatis and Mycobacterium tuberculosis We show here that the ripA and ripB genes are individually dispensable in M. smegmatis and that at least one of the genes must be expressed for viability. We characterized strains carrying in-frame deletion mutations of ripA and ripB and found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ΔripA mutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ΔripD and ΔripAΔripD mutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss of ripD in the ΔripA background partially rescued the antibiotic and lysozyme phenotypes of the ΔripA mutant. IMPORTANCE: We show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD.

One of these is not like the others.

A Mycobacterium tuberculosis metA mutant that is auxotrophic for methionine is unlike other auxotrophic mutants of this important species as methionine starvation results in rapid death instead of cessation of growth. Evidence suggests that this phenotype results from starvation affecting essential pathways that utilize S-adenosylmethionine in addition to methionine.

Activation of the aryl hydrocarbon receptor during development enhances the pulmonary CD4+ T-cell response to viral infection.

Respiratory infections are a threat to health and economies worldwide, yet the basis for striking variation in the severity of infection is not completely understood. Environmental exposures during development are associated with increased severity and incidence of respiratory infection later in life. Many of these exposures include ligands of the aryl hydrocarbon receptor (AHR), a transcription factor expressed by immune and nonimmune cells. In adult animals, AHR activation alters CD4(+) T cells and changes immunopathology. Developmental AHR activation impacts CD4(+) T-cell responses in lymphoid tissues, but whether skewed responses are also present in the infected lung is unknown. To determine whether pulmonary CD4(+) T-cell responses are modified by developmental AHR activation, mice were exposed to the prototypical AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin during development and infected with influenza virus as adults. Lungs of exposed offspring had greater bronchopulmonary inflammation compared with controls, and activated, virus-specific CD4(+) T cells contributed to the infiltrating leukocytes. These effects were CD4(+) T cell subset specific, with increases in T helper type 1 and regulatory T cells, but no change in the frequency of T helper type 17 cells in the infected lung. This is in direct contrast to prior reports of suppressed conventional CD4(+) T-cell responses in the lymph node. Using adoptive transfers and manipulating the pathogen properties, we determined that developmental exposure influenced factors intrinsic and extrinsic to CD4(+) T cells and may involve developmentally induced changes in signals from infected lung epithelial cells. Thus developmental exposures lead to context-dependent changes in pulmonary CD4(+) T-cell subsets, which may contribute to differential responses to respiratory infection.

Revisiting the Gram-negative lipoprotein paradigm.

UNLABELLED: The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS Δlnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the Δlnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. IMPORTANCE: This paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria.

Structure-function analysis of VapB4 antitoxin identifies critical features of a minimal VapC4 toxin-binding module.

UNLABELLED: Bacterial toxin-antitoxin systems play a critical role in the regulation of gene expression, leading to developmental changes, reversible dormancy, and cell death. Type II toxin-antitoxin pairs, composed of protein toxins and antitoxins, exist in nearly all bacteria and are classified into six groups on the basis of the structure of the toxins. The VapBC group comprises the most common type II system and, like other toxin-antitoxin systems, functions to elicit dormancy by inhibiting protein synthesis. Activation of toxin function requires protease degradation of the VapB antitoxin, which frees the VapC toxin from the VapBC complex, allowing it to hydrolyze the RNAs required for translation. Generally, type II antitoxins bind with high specificity to their cognate toxins via a toxin-binding domain and endow the complex with DNA-binding specificity via a DNA-binding domain. Despite the ubiquity of VapBC systems and their critical role in the regulation of gene expression, few functional studies have addressed the details of VapB-VapC interactions. Here we report on the results of experiments designed to identify molecular determinants of the specificity of the Mycobacterium tuberculosis VapB4 antitoxin for its cognate VapC4 toxin. The results identify the minimal domain of VapB4 required for this interaction as well as the amino acid side chains required for binding to VapC4. These findings have important implications for the evolution of VapBC toxin-antitoxin systems and their potential as targets of small-molecule protein-protein interaction inhibitors. IMPORTANCE: VapBC toxin-antitoxin pairs are the most widespread type II toxin-antitoxin systems in bacteria, where they are thought to play key roles in stress-induced dormancy and the formation of persisters. The VapB antitoxins are critical to these processes because they inhibit the activity of the toxins and provide the DNA-binding specificity that controls the synthesis of both proteins. Despite the importance of VapB antitoxins and the existence of several VapBC crystal structures, little is known about their functional features in vivo. Here we report the findings of the first comprehensive structure-function analysis of a VapB toxin. The results identify the minimal toxin-binding domain, its modular antitoxin function, and the specific amino acid side chains required for its activity.

Genetic characterization of mycobacterial L,D-transpeptidases.

l,d-Transpeptidases (Ldts) catalyse the formation of 3-3 cross-links in peptidoglycans (PGs); however, the role of these enzymes in cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage of 3-3 cross-links (~30-80 %) than the PG in most other bacteria, suggesting that they are particularly important to mycobacterial cell wall biology. The genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis encode multiple Ldt genes, but it is not clear if they are redundant. We compared the sequences of the Ldt proteins from 18 mycobacterial genomes and found that they can be grouped into six classes. We then constructed M. smegmatis strains lacking single or multiple Ldt genes to determine the physiological consequence of the loss of these enzymes. We report that of the single mutants, only one, ΔldtC (MSMEG_0929, class 5), displayed an increased susceptibility to imipenem - a carbapenem antibiotic that inhibits the Ldt enzymes. The invariant cysteine in the active site of LdtC was required for function, consistent with its role as an Ldt. A triple mutant missing ldtC and both of the class 2 genes displayed hypersusceptibility to antibiotics, lysozyme and d-methionine, and had an altered cellular morphology. These data demonstrated that the distinct classes of mycobacterial Ldts may reflect different, non-redundant functions and that the class 5 Ldt was peculiar in that its loss, alone and with the class 2 proteins, had the most profound effect on phenotype.

Genetics of Peptidoglycan Biosynthesis.

The complex cell envelope is a hallmark of mycobacteria and is anchored by the peptidoglycan layer, which is similar to that of Escherichia coli and a number of other bacteria but with modifications to the monomeric units and other structural complexities that are likely related to a role for the peptidoglycan in stabilizing the mycolyl-arabinogalactan-peptidoglycan complex (MAPc). In this article, we will review the genetics of several aspects of peptidoglycan biosynthesis in mycobacteria, including the production of monomeric precursors in the cytoplasm, assembly of the monomers into the mature wall, cell wall turnover, and cell division. Finally, we will touch upon the resistance of mycobacteria to ß-lactam antibiotics, an important class of drugs that, until recently, have not been extensively exploited as potential antimycobacterial agents. We will also note areas of research where there are still unanswered questions.

Phenotypic analysis of Eschericia coli mutants lacking L,D-transpeptidases.

Escherichia coli has five genes encoding L,D-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun's lipoprotein to the peptidoglycan (PG), while the other two (YnhG, YcbB) form direct meso-diaminopimelate (DAP-DAP, or 3-3) cross-links within the PG. In addition, Ldt enzymes can also incorporate non-canonical D-amino acids, such as D-methionine, into the PG. To further investigate the role of these enzymes and, in particular, 3-3 linkages in cell envelope physiology we constructed and phenotypically characterized a variety of multiple Ldt deletion mutants of E. coli. We report that a triple deletion mutant lacking ybiS, erfK and ycfS is hypersusceptible to the metal-chelating agent EDTA, leaks periplasmic proteins and is resistant to the toxic effect of D-methionine. A double ynhG ycbB mutant had no discernible phenotype; however, examination of the phenotypes of various Ldt mutants bearing an additional DAP auxotrophic mutation (dapA : : Cm) showed that a quintuple mutant strain lacking all Ldt genes was severely impaired for growth on media with limited DAP. These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of outer membrane stability while loss of the Ldt enzymes involved with DAP-DAP linkages had no observable effect on the cell envelope. Loss of all Ldt enzymes proved detrimental to growth when cells were starved for DAP, indicating a combined role for both 3-3 and Braun's lipoprotein cross-links in cell viability only under a specific PG stress.

Updating and curating metabolic pathways of TB.

The sequencing of complete genomes has accelerated biomedical research by providing information about the overall coding capacity of bacterial chromosomes. The original TB annotation resulted in putative functional assignment of ∼60% of the genes to specific metabolic functions, however, the other 40% of the encoded ORFs where annotated as conserved hypothetical proteins, hypothetical proteins or encoding proteins of unknown function. The TB research community is now at the beginning of the next phases of post-genomics; namely reannotation and functional characterization by targeted experimentation. Arguably, this is the most significant time for basic microbiology in recent history. To foster basic TB research, the Tuberculosis Community Annotation Project (TBCAP) jamboree exercise began the reannotation effort by providing additional information for previous annotations, and refining and substantiating the functional assignment of ORFs and genes within metabolic pathways. The overall goal of the TBCAP 2012 exercise was to gather and compile various data types and use this information with oversight from the scientific community to provide additional information to support the functional annotations of encoding genes. Another objective of this effort was to standardize the publicly accessible Mycobacterium tuberculosis reference sequence and its annotation. The greatest benefit of functional annotation information of genome sequence is that it fuels TB research for drug discovery, diagnostics, vaccine development and epidemiology.

Mycobacteriophage Ms6 LysA: a peptidoglycan amidase and a useful analytical tool.

Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

TetR-based gene regulation systems for Francisella tularensis.

There are a number of genetic tools available for studying Francisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for F. tularensis based on the Tet repressor, TetR. For the inducible system, a tet operator sequence was cloned into a modified F. tularensis groESL promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon, luxCDABE, was cloned under the hybrid Francisella tetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with tetO. Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml(-1). In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of FTRp regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the lux reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring F. tularensis protein function.