Carbapenem-resistant (CRE) are rapidly spreading and taking a staggering toll on all health care systems, largely due to the dissemination of genes coding for potent carbapenemases. An important family of carbapenemases are the Zn(II)-dependent β-lactamases, known as metallo-β-lactamases (MBLs). Among them, the New Delhi metallo-β-lactamase (NDM) has experienced the fastest and widest geographical spread. While other clinically important MBLs are soluble periplasmic enzymes, NDMs are lipoproteins anchored to the outer membrane in Gram-negative bacteria. This unique cellular localization endows NDMs with enhanced stability upon the Zn(II) starvation elicited by the immune system response at the sites of infection. Since the first report of NDM-1, new allelic variants (16 in total) have been identified in clinical isolates differing by a limited number of substitutions. Here, we show that these variants have evolved by accumulating mutations that enhance their stability or the Zn(II) binding affinity, overriding the most common evolutionary pressure acting on catalytic efficiency. We identified the ubiquitous substitution M154L as responsible for improving the Zn(II) binding capabilities of the NDM variants. These results also reveal that Zn(II) deprivation imposes a strict constraint on the evolution of this MBL, overriding the most common pressures acting on catalytic performance, and shed light on possible inhibitory strategies.

Salmonella enterica success on infection and transmission between hosts relies greatly on its ability to recognize, to evade, and to even exploit host defenses on its favor (Behnsen et al., 2015). Critical to Salmonella pathogenesis is the coordinated assembly of two type III secretion systems (T3SS) that are located at specific genome regions called pathogenicity islands (SPIs). The Salmonella pathogenicity island SPI-1 is associated with the invasion of epithelial cells, whereas SPI-2 is required for intracellular survival and proliferation (Agbor &

Cellulose is a major component of the Salmonella biofilm extracellular matrix and it is considered an antivirulence factor because it interferes with Salmonella survival inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm extracellular matrix formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work we identified a SPI-2 encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, STM1389, also known as orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage survival. An increased expression of csgD is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase coding-gene, bcsA, restored intramacrophage survival to rates comparable to wild type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and in consequence activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.