Prior studies suggest that the impaired healing seen in diabetic wounds derives from a state of persistent hyper-inflammation characterized by harmful increases in inflammatory leukocytes including macrophages. However, such studies have focused on wounds at later time points (day 10 or older), and very little attention has been given to the dynamics of macrophage responses in diabetic wounds early after injury. Given the importance of macrophages for the process of healing, we studied the dynamics of macrophage response during early and late phases of healing in diabetic wounds. Here, we report that early after injury, the diabetic wound exhibits a significant delay in macrophage infiltration. The delay in the macrophage response in diabetic wounds results from reduced Chemokine (C-C motif) ligand 2 (CCL2) expression. Importantly, one-time treatment with chemoattractant CCL2 significantly stimulated healing in diabetic wounds by restoring the macrophage response. Our data demonstrate that, rather than a hyper-inflammatory state; the early diabetic wound exhibits a paradoxical and damaging decrease in essential macrophage response. Our studies suggest that the restoration of the proper kinetics of macrophage response may be able to jumpstart subsequent healing stages. CCL2 chemokine-based therapy may be an attractive strategy to promote healing in diabetic wounds.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

SummaryType III secreted (T3SS) effectors are important virulence factors in acute infections caused by Pseudomonas aeruginosa. PA103, a well-studied human lung isolate, encodes and secretes two effectors, ExoU and ExoT. ExoU is a potent cytotoxin that causes necrotic cell death. In addition, PA103 can induce cell death in macrophages in an ExoUindependent but T3SS-dependent manner. We now demonstrate that ExoT is both necessary and sufficient to cause apoptosis in HeLa cells and that it activates the mitochondrial/cytochrome c-dependent apoptotic pathway. We further show that ExoT induction of cell death is primarily dependent on its ADP ribosyltransferase domain activity. Our data also indicate that the T3SS apparatus can cause necrotic cell death, which is effectively blocked by ExoT, suggesting that P. aeruginosa may have evolved strategies to prevent T3SS-induced necrosis.

The expression of many gene products required during the early stages of Bacillus subtilis sporulation is regulated by sinIR operon proteins. Transcription of sinIR from the P1 promoter is induced at the end of exponential growth. In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A. In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are trans-acting transcriptional factors that bind to the P1 promoter region of sinIR. We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor, A , form of RNA polymerase.Natural environments are oligotrophic (35). Organisms such as the common soil bacterium Bacillus subtilis frequently exist in slow-or nongrowing physiological states. The rich diversity of B. subtilis transition-state regulatory systems (50,55,56) confirms the biological importance of managing the transition from rapid-to slow-to nongrowing cell states. Depending on the environmental cues present, B. subtilis transition-state regulation can channel a cell toward motility, nutrient scavenging through the production of extracellular enzymes, competence, or sporulation cell fates (for a review, see references 12 and 53). The best-characterized B. subtilis transition-state regulators are the AbrB, Hpr, Spo0A, and SinR DNA-binding proteins.Recent structural studies have shown that the AbrB protein is a tetramer of 10,500-Da subunits that interacts with a variety of specific nucleotide sequences, presumably by recognizing a particular three-dimensional DNA architecture (54,59,62). AbrB can function as a repressor of genes such as spo0E, spo0H, spoVG, and aprE (14,34,43,64) and as an activator of genes such as hpr and the rbs operon (52, 53). Transcription of abrB is controlled by negative autoregulation and repression by Spo0A (53, 55). The hpr gene product is a 23,718-Da protein, which was originally identified as a locus (hpr, scoC, and catA) for mutations causing protease overproduction and cataboliteresistant sporulation (10,21,39). Hpr binds to a consensus DNA sequence RATAnTATY (25,53). Hpr represses the expression of the protease genes aprE and nprE and oligopeptide permease operons (20,26) and when present on a multicopy plasmid can inhibit sporulation in an as-yet-undetermined manner (39). The Spo0A 29,691-Da protein is the master controller of early developmental events (55, 56). Metabolic and environmental signals cause the autophosphorylation of sensor kinases such as KinA, KinB, and KinC (1, 24, 27, 41), which transfer phosphate groups through a phosphorelay (Spo0F and Spo0B) to generate Spo0AϳP (24, 55, 57). Spo0AϳP recognizes a 0A box DNA sequence, TGNCGAA (51). Spo0AϳP is a repressor of abrB transcription and an activator of spoIIA, spoIIG, and spoIIE operon expression (4,45,51,57,63). spo0A expression is controlled by A and H promoters (9, 44, 49). Vegetative spo0A expression originates from the A promoter, and cataboli...

Pseudomonas aeruginosa is an opportunistic pathogen that requires preexisiting epithelial injury to cause acute infections. We report that P. aeruginosa inhibits mammalian cytokinesis in a type III secretion system and exotoxin T (ExoT)-dependent manner. ExoT is a bifunctional type III secretion system effector protein that contains an N-terminal GTPase-activating protein domain and a C-terminal ADP-ribosyl transferase domain. Each of its domains inhibits cytokinesis in a kinetically, morphologically, and mechanistically distinct manner. The GTPase-activating protein-mediated inhibition of cytokinesis occurs early, likely as a consequence of its inhibitory effect on RhoA. The ADP-ribosyl transferase domain inhibits late steps of cytokinesis by blocking syntaxin-2 localization to the midbody, an event essential for completion of cytokinesis. These findings provide an example of a bacterial pathogen targeting cytokinesis.type III secretion system ͉ virulence factor ͉ microbial pathogenesis ͉ wound healing