Investigation of the Role of Genes Encoding Zinc Exporters zntA, zitB, and fieF during Salmonella Typhimurium Infection.

The transition metal zinc is involved in crucial biological processes in all living organisms and is essential for survival of Salmonella in the host. However, little is known about the role of genes encoding zinc efflux transporters during Salmonella infection. In this study, we constructed deletion mutants for genes encoding zinc exporters (zntA, zitB, and fieF) in the wild-type (WT) strain Salmonella enterica serovar Typhimurium (S. Typhimurium) 4/74. The mutants 4/74ΔzntA and 4/74ΔzntA/zitB exhibited a dramatic growth delay and abrogated growth ability, respectively, in Luria Bertani medium supplemented with 0.25 mM ZnCl2 or 1.5 mM CuSO4 compared to the WT strain. In order to investigate the role of genes encoding zinc exporters on survival of S. Typhimurium inside cells, amoeba and macrophage infection models were used. No significant differences in uptake or survival were detected for any of the mutants compared to the WT during infection of amoebae. In natural resistance-associated macrophage protein 1 (Nramp1)-negative J774.1 murine macrophages, significantly higher bacterial counts were observed for the mutant strains 4/74ΔzntA and 4/74ΔzntA/zitB compared to the WT at 4 h post-infection although the fold net replication was similar between all the strains. All four tested mutants (4/74ΔzntA, 4/74ΔzitB, 4/74ΔfieF, and 4/74ΔzntA/zitB) showed enhanced intracellular survival capacity within the modified Nramp1-positive murine RAW264.7 macrophages at 20 h post-infection. The fold net replication was also significantly higher for 4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB mutants compared to the WT. Intriguingly, the ability to survive and cause infection was significantly impaired in all the three mutants tested (4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB) in C3H/HeN mice, particularly the double mutant 4/74ΔzntA/zitB was severely attenuated compared to the WT in all the three organs analyzed. These findings suggest that these genes encoding zinc exporters, especially zntA, contribute to the resistance of S. Typhimurium to zinc and copper stresses during infection.

Effects of Listeria monocytogenes EGD-e and Salmonella enterica ser. Typhimurium LT2 chitinases on intracellular survival in Dictyostelium discoideum and mammalian cell lines.

Some bacterial pathogens produce chitinases as virulence factors during host infection. The molecular target of such enzymes in non-chitinous hosts remains uncertain. We studied the importance of Listeria monocytogenes EGD-e and Salmonella enterica ser. Typhimurium LT2 chitinases for intracellular survival in Dictyostelium discoideum, and for Salmonella, also infection of mammalian cell lines, and a mouse model. The Salmonella chitinase did not contribute significantly to infection of D. discoideum, mammalian cell lines or mice. However, survival in D. discoideum was clearly reduced for Listeria mutants deficient of ChiB (8-fold) or deficient of both ChiA and ChiB (22-fold). Our findings suggest that chitinases from the two species play different roles in virulence.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galß1-4GlcNAc) and LacdiNAc (GalNAcß1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

Bacterial chitinases and chitin-binding proteins as virulence factors.

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.