Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

New Insights into the Role of Zinc Acquisition and Zinc Tolerance in Group A Streptococcal Infection.

Zinc plays an important role in host innate immune function. However, the innate immune system also utilizes zinc starvation ("nutritional immunity") to combat infections. Here, we investigate the role of zinc import and export in the protection of Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive bacterial pathogen responsible for a wide spectrum of human diseases, against challenge from host innate immune defense. In order to determine the role of GAS zinc import and export during infection, we utilized zinc import (ΔadcA ΔadcAII) and export (ΔczcD) deletion mutants in competition with the wild type in both in vitro and in vivo virulence models. We demonstrate that nutritional immunity is deployed extracellularly, while zinc toxicity is utilized upon phagocytosis of GAS by neutrophils. We also show that lysosomes and azurophilic granules in neutrophils contain zinc stores for use against intracellular pathogens.

ZrgA contributes to zinc acquisition in Vibrio parahaemolyticus.

Metals are nutrients essential for almost all lifeforms. Bacteria have evolved several mechanisms to overcome the metal restrictions imposed by the host. Vibrio parahaemolyticus causes severe threats to public health and significant economic losses in shrimp aquaculture. Herein, we report that ZrgA contributes to zinc acquisition in this pathogen. The operon VP_RS01455 to VP_RS01475 of V. parahaemolyticus encodes the putative Zn transporter ZrgABCDE, whose homologs are widely distributed in Vibrionaceae. RNA sequencing analysis revealed that V. parahaemolyticus modulates the transcriptome in response to Zn limitation. Genes in the Zinc uptake regulator (Zur) regulon are upregulated during Zn limitation, including three genes annotated to encode Zn-binding proteins. Significant upregulation of these three genes during Zn limitation was also confirmed by quantitative real-time PCR (qRT-PCR) analysis. However, only the mutants containing a VP_RS01470 (zrgA) deletion exhibited impaired growth under Zn-deficient conditions, indicating that VP_RS01470 plays the predominant role in V. parahaemolyticus Zn acquisition. The VP_RS01470 deletion mutant displayed a false appearance of decreased swimming motility under Zn-deficient conditions, as revealed by the fact that the polar flagellar-related genes were not downregulated in the mutant. Moreover, VP_RS01470 deletion produced no noticeable impact on the swarming motility and virulence in mice. qRT-PCR analysis and ß-galactosidase activity assays indicated that Zur negatively regulates VP_RS01470 expression in V. parahaemolyticus. Collectively, our findings suggest that ZrgA is required for Zn acquisition in V. parahaemolyticus and highlight the importance of detecting the expression of flagellar genes during analysis of motility of a mutant deficient in growth.

The AdcR-regulated AdcA and AdcAII contribute additively to zinc acquisition and virulence in Streptococcus suis.

Metals are necessary elements for bacteria. Typically, vertebrate hosts restrict invading bacterial pathogens from accessing metals. Therefore, bacteria have evolved high-affinity metal importers to acquire metals. Streptococcus suis is a major swine pathogen and an emerging zoonotic agent that endangers the swine industry and human health worldwide. Herein, we aimed to identify the zinc acquisition systems in S. suis and evaluate their roles in bacterial virulence. Bioinformatic analyses revealed that S. suis encodes homologues of AdcA and AdcAII, two well-characterised Zn-binding lipoproteins in certain streptococci. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that the expressions of adcA and adcAII were significantly upregulated in response to Zn limitation, with a higher expression level of adcAII than adcA. Gene deletion mutants and complementation strains were constructed; their growth characteristics under Zn-deficient and Zn-replete conditions indicated that AdcA and AdcAII have overlapping functionality in Zn acquisition. A mouse infection model was used to evaluate the roles of AdcA and AdcAII in S. suis virulence. Mice infected with the double mutant ΔadcAΔadcAII exhibited a significantly higher survival rate, decreased bacterial burden, and lower production of inflammatory cytokines compared to those infected with the wild type (WT) strain. Furthermore, ΔadcAΔadcAII showed reduced competitiveness in infection establishment compared with the WT strain. RNA sequencing, qRT-PCR, and electrophoretic mobility shift assays revealed that AdcR negatively regulates the expressions of adcA and adcAII. Collectively, our results demonstrated that AdcA and AdcAII, which are negatively regulated by AdcR, contribute additively to zinc acquisition and virulence in S. suis.

The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis.

Zinc homeostasis is crucial for the development and stress resistance of bacteria in the environment. Serial zinc sensing transcriptional regulators, zinc transporters and zinc binding proteins were found to maintain the zinc homeostasis in bacteria. Zur is a zinc uptake regulator that is widely distributed in species, and ZnuABC, as well as the Type VI Secretion System (T6SS4) function in zinc acquisition. Here, we report that the regulator Zur inhibits the expression of the ZnuABC which inhibition could be eliminated at low zinc level, and upregulates the T6SS4 operon in Yersinia pseudotuberculosis to facilitate Zn2+ uptake and oxidative stress resistance. Zur regulates the expression of ZnuABC and T6SS4 by directly binding to their promoter regions. Zur senses the Zn2+ concentration and represses ZnuABC in a Zn2+-containing environment. Zur works as an auxiliary regular activator of T6SS4, facilitating oxidative stress resistance. This study revealed the dual function of regulator Zur on ZnuABC and T6SS4, and enriched the knowledge of Zn2+ homeostasis maintenance in Y. pseudotuberculosis.

Structural characterisation of the HT3 motif of the polyhistidine triad protein D from Streptococcus pneumoniae.

The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition.