Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria.

Impaired wound closure is a growing medical problem associated with metabolic diseases and aging. Immune cells play important roles in wound healing by following instructions from the microenvironment. Here, we developed a technology to bioengineer the wound microenvironment and enhance healing abilities of the immune cells. This resulted in strongly accelerated wound healing and was achieved by transforming Lactobacilli with a plasmid encoding CXCL12. CXCL12-delivering bacteria administrated topically to wounds in mice efficiently enhanced wound closure by increasing proliferation of dermal cells and macrophages, and led to increased TGF-ß expression in macrophages. Bacteria-produced lactic acid reduced the local pH, which inhibited the peptidase CD26 and consequently enhanced the availability of bioactive CXCL12. Importantly, treatment with CXCL12-delivering Lactobacilli also improved wound closure in mice with hyperglycemia or peripheral ischemia, conditions associated with chronic wounds, and in a human skin wound model. Further, initial safety studies demonstrated that the topically applied transformed bacteria exerted effects restricted to the wound, as neither bacteria nor the chemokine produced could be detected in systemic circulation. Development of drugs accelerating wound healing is limited by the proteolytic nature of wounds. Our technology overcomes this by on-site chemokine production and reduced degradation, which together ensure prolonged chemokine bioavailability that instructed local immune cells and enhanced wound healing.

Antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defence peptides.

BACKGROUND: The clinical development of antimicrobial peptides (AMPs) is currently under evaluation to combat the rapid increase in MDR bacterial pathogens. However, many AMPs closely resemble components of the human innate immune system and the ramifications of prolonged bacterial exposure to AMPs are not fully understood. OBJECTIVES: We show that in vitro serial passage of a clinical USA300 MRSA strain in a host-mimicking environment containing host-derived AMPs results in the selection of stable AMP resistance. METHODS: Serial passage experiments were conducted using steadily increasing concentrations of LL-37, PR-39 or wheat germ histones. WGS and proteomic analysis by MS were used to identify the molecular mechanism associated with increased tolerance of AMPs. AMP-resistant mutants were characterized by measuring in vitro fitness, AMP and antibiotic susceptibility, and virulence in a mouse model of sepsis. RESULTS: AMP-resistant Staphylococcus aureus mutants often displayed little to no fitness cost and caused invasive disease in mice. Further, this phenotype coincided with diminished susceptibility to both clinically prescribed antibiotics and human defence peptides. CONCLUSIONS: These findings suggest that therapeutic use of AMPs could select for virulent mutants with cross-resistance to human innate immunity as well as antibiotic therapy. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.

Fitness of Salmonella mutants resistant to antimicrobial peptides.

OBJECTIVES: To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. METHODS: Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. RESULTS: Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. CONCLUSIONS: Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored.

Screening and Development of New Inhibitors of FtsZ from M. Tuberculosis.

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics.

Genetic evidence that mycobacterial FtsZ and FtsW proteins interact, and colocalize to the division site in Mycobacterium smegmatis.

We provide genetic evidence to show that the Mycobacterium tuberculosis FtsZ and FtsW proteins interact, and that these interactions are biologically relevant. Furthermore, we show by fluorescence microscopy that Mycobacterium smegmatis FtsW is part of its septasomal complex and colocalizes with FtsZ to the midcell sites. Colocalization experiments reveal that approximately 27% of the cells with septal Z-rings contain FtsW whereas 93% of the cells with FtsW bands are associated with FtsZ indicating that FtsW is late recruit to the septum, as in Escherichia coli. Our results suggest that mycobacterial FtsZ can localize to the septum independent of FtsW, and that interactions of FtsW with FtsZ are critical for the formation of productive FtsZ-rings and the cell division process in mycobacteria.

Mechanisms and fitness costs of resistance to antimicrobial peptides LL-37, CNY100HL and wheat germ histones.

Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.

ChiZ levels modulate cell division process in mycobacteria.

We have previously shown that expression of chiZ (Rv2719c), encoding a cell wall hydrolase, is upregulated in response to DNA damaging agents and exposure to cephalexin. Furthermore, increased levels of ChiZ lead to decreased viability, loss of membrane integrity and defects in FtsZ-GFP localization and cell division. We now show that ChiZ N'-terminal 110 amino acid region, containing the cell wall hydrolase activity, is sufficient to modulate FtsZ-GFP localization. Further, we found that FtsZ-GFP rings are stabilized in a chiZ deletion strain indicating that ChiZ activity regulates FtsZ assembly. Overexpression of ftsZ did not reverse the reduction in viability caused by overproduction of ChiZ indicating that ChiZ neither interacts with nor directly influences FtsZ assembly. Bacterial two-hybrid assays revealed that ChiZ interacts with FtsI and FtsQ, two other septasomal proteins, but not with FtsZ. Finally, we show that ChiZ is not required for virulence of Mycobacterium tuberculosis in murine macrophages and mice. Our data suggest that optimal levels and activity of the cell wall hydrolase ChiZ are required for regulated cell division in mycobacteria.

Mycobacterium tuberculosis ftsZ expression and minimal promoter activity.

Optimal levels of ftsZ gene product are shown to be required for initiation of the cell division process in Mycobacterium tuberculosis. Here, we report that the ftsZ gene expression is sharply down-regulated during starvation and hypoxia, conditions that are believed to result in growth arrest, but is restored upon dilution of cultures into fresh oxygen-rich media. Primer extension analysis identified four transcriptional start sites, designated as P1, P2, P3 and P4 at nucleotide positions -43, -101, -263, and -787, respectively, in the immediate upstream flanking region of the ftsZ initiation codon. Promoter deletion and homologous recombination experiments revealed that ftsZ expression from the 101-bp region is sufficient for M. tuberculosis viability. All promoter strains had reduced FtsZ levels compared to wild-type, although the loss of P4 severely compromised FtsZ levels during both the active and stationary phases. We propose that ftsZ expression from all promoters is required for optimal intracellular FtsZ levels and that the activities of P4 and possibly other promoters are down-regulated during growth-arrest conditions.

Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase.

The genetic factors responsible for the regulation of cell division in Mycobacterium tuberculosis are largely unknown. We showed that exposure of M. tuberculosis to DNA damaging agents, or to cephalexin, or growth of M. tuberculosis in macrophages increased cell length and sharply elevated the expression of Rv2719c, a LexA-controlled gene. Overexpression of Rv2719c in the absence of DNA damage or of antibiotic treatment also led to filamentation and reduction in viability both in broth and in macrophages indicating a correlation between Rv2719c levels and cell division. Overproduction of Rv2719c compromised midcell localization of FtsZ rings, but had no effect on the intracellular levels of FtsZ. In vitro, the Rv2719c protein did not interfere with the GTP-dependent polymerization activity of FtsZ indicating that the effects of Rv2719c on Z-ring assembly are indirect. Rv2719c protein exhibited mycobacterial murein hydrolase activity that was localized to the N-terminal 110 amino acids. Visualization of nascent peptidoglycan (PG) synthesis zones by probing with fluoresceinated vancomycin (Van-FL) and localization of green fluorescent protein-Rv2719c fusion suggested that the Rv2719c activity is targeted to potential PG synthesis zones. We propose that Rv2719c is a potential regulator of M. tuberculosis cell division and that its levels, and possibly activities, are modulated under a variety of growth conditions including growth in vivo and during DNA damage, so that the assembly of FtsZ-rings, and therefore the cell division, can proceed in a regulated manner.

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings.

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo.

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.