The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn 2+ and Zn 2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn 2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn 2+ -binding site from six histidine residues, which distinguishes CP from all other Mn 2+ -binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn 2+ and Zn 2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn 2+ sequestration. These data establish the importance of Mn 2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.bacterial pathogenesis | Staphylococcus aureus | antibiotic resistance | protein crystal structure | isothermal titration calorimetry B acterial pathogens are a significant threat to global public health. This threat is compounded by the fact that these organisms are rapidly becoming resistant to all relevant antimicrobials. Of particular note is the recent emergence of antibiotic-resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States (1) and arguably the most important threat to the public health of the developed world. Consequently, the identification of therapeutics to treat bacterial pathogens is paramount to our continued ability to limit this infectious threat.One promising area of potential therapeutic development involves targeting bacterial access to essential transition metals. This strategy is based on the fact that all bacterial pathogens require these nutrient metals to colonize their hosts (2-5). In vertebrates, the bacterial need for nutrient transition metals is counteracted by the sequestration of these metals by the host. This limitation of essential nutrients, termed "nutritional immunity," is a potent defense against infection (6). Although a variety of metals is required for microbial growth, studies of nutritional immunity have been primarily restricted to the struggle for iron (Fe) between host and pathogen (7-9).An innate immune factor, calprotectin (CP), is abundant in neutrophils and plays a key role in nutritional immunity. CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10-14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11, 13). Moreover, CP-deficient mice have increased microbial burdens following systemic challenge, und...

Acinetobacter baumannii is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against A. baumannii was investigated. These experiments revealed that CP inhibits A. baumannii growth in vitro through chelation of Mn and Zn. Consistent with these in vitro data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an A. baumannii Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in A. baumannii. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant A. baumannii underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant A. baumannii infections.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA.

WEE1 kinase is a key regulator of the G2/M transition. The WEE1 kinase inhibitor AZD1775 (WEE1i) induces origin firing in replicating cells. We show that WEE1i induces CDK1-dependent RIF1 phosphorylation and CDK2- and CDC7-dependent activation of the replicative helicase. WEE1 suppresses CDK1 and CDK2 kinase activities to regulate the G1/S transition after the origin licensing is complete. We identify a role for WEE1 in cell cycle regulation and important effects of AZD1775, which is in clinical trials.