Project title:

Optimal dietary metal ion uptake and its role in protection against childhood bacterial disease

Chief Investigator:

Dr Bart Eijkelkamp

Funding Amount:

$35,000

Recipient:

The University of Adelaide

Overview:

Nearly 2 billion people worldwide have a poor zinc status and in the Western world childhood zinc deficiency is on the increase due to unhealthy diets and obesity. The immune system utilizes zinc and copper as antimicrobials to combat infectious diseases. This places children with a poor zinc status at increased risk of infections by widely abundant childhood respiratory pathogens such as Streptococcus pneumoniae. Although zinc deficiency has previously been associated with increased disease burden, the role of zinc and copper as antimicrobials during infection remains unknown.

Research outcomes:

Researchers:

Bart Eijkelkamp, Christopher McDevitt

Research Completed:

2016

Research Findings:

The project by Cl Eijkelkamp & Al McDevitt investigated the role of metals in bacterial infection control. It focussed on how the innate immune system uses these metal ions in an attempt to restrict bacterial proliferation. The application of their murine zinc-restriction model in examining the role of zinc during bacterial infection has led to various exciting novel findings. Primarily, the study has revealed that the host modulates the zinc abundance to restrict growth by the invading pathogen. In the blood, zinc availability is restricted, hence bacteria utilise high-affinity zinc import systems for their survival. In contrast, bacteria rely upon the active efflux of zinc for persistence in lung tissues. The study also revealed an exciting role for zinc in the killing of bacteria within macrophages and neutrophils1 the two most significant phagocytic cells in the fight against Infectious diseases. Overall, this study has provided key insights into the molecular basis behind zinc supplementation of children highly susceptible to bacteria l Infections.

Key Outcomes:

Optimal dietary metal ion uptake and its role in protection against childhood bacterial disease
To study the impact of dietary zinc deficiency and how zinc supplementation can aid in protection against bacterial infections, we developed a mouse model in which tissue zinc levels can be increased by dietary supplementation. The serum zinc levels in our model reflect that seen in low zinc groups in the Western countries, such as obese children, who are at greatly increased risk of enteric and respiratory infections. In addition to studying the role of zinc in nutritional immunity, copper is also of growing importance in nutritional immunity and antimicrobial responses. Our recent analyses of host metal ions have revealed that upon infection with the major child hood pathogen Streptococcus pneumoniae (pneumococcus), copper levels increase >10-fold in various niches. Although zinc and copper are both known to play a key role in killing invading bacteria, the molecular bases of how these essential metal ions target bacterial pathogens, either individually or in concert, remain to be determined.

AI M 1: To examine the effect of zinc and copper on bacterial infections
The findings from Aim 1 have provided comprehensive insights into the role of zinc in restricting bacterial colonization in a murine model of infection. We have identified highly niche-specific roles for zinc, as restriction of zinc availability appears to be the mode of restricting bacterial proliferation in the blood, whereas in the lung1 the host redirects zinc towards to invading bacterium to impair its fitness. Interestingly, no differences were observed between the two mouse populations in the pleural cavity, in terms of metal abundance or pneumococcal burden. Hence, this study has, for the first time, revealed an inverse correlation between zinc abundance and pneumococcal burden.

To ascertain if host metal abundance has a direct effect on the pneumococcus, we examined the transcription levels of the major metal homeostasis mechanisms in pneumococci isolated from the zinc-limited or zinc-replete mice. These highly innovative data revealed that the pneumococcus is directly affected by metal abundance in the host environment as the zinc efflux system (czcD) was significantly upregulated in pneumococci isolated from the blood or lungs of zinc­replete mice as compared to those isolated from the zinc-limited mice. In contrast, expression of phtD, a marker for zinc acquisition, was lower in pneumococci isolated from the blood or lungs of zinc-replete mice as compared to those isolated from zinc-limited mice. The transcriptional profiling of czcD and phtD in pneumococci isolated from the pleura l cavity revealed no transcriptional differences1 which highlights that the metal homeostasis of pneumococci is specifically affected by environmental metal abundance.

In previous in vitro analyses, we have shown that zinc stress causes manganese starvation in the pneumococcus. However, how zinc stress affects the pneumococcus during infection remained unknown. Hence, we examined the transcription of psaA, a critical component for manganese acquisition, in pneumococci isolated from the lung1 blood1 and pleural cavity of zinc-restricted and zinc-replete mice. This cutting-edge research showed that pneumococci are depleted of manganese when zinc is in high abundance, thereby high lighting the clinical relevance of our previous in vitro studies.

Our analyses of a pneumococcal copper efflux mutant revealed that copper intoxication is important mode of combat against pneumococci in the lungs. However, these data also revealed for the first time1 that within this niche, zinc and copper stress are not used as antimicrobials in concert, despite their significant antimicrobial synergy.

AI M 2: To determine the antimicrobial mechanisms of zinc and copper
We have performed extensive transcriptomic and metabolic analyses of zinc and copper stress in the pneumococcus, which have revealed the far-reaching effects of metal stress on bacterial physiology. Our data has revealed how a metal imbalance as a result of zinc or copper stress, leads to dysfunctionality of various metal-dependent proteins, such as those involved in capsule biosynthesis. We are currently further investigating the metal-binding properties of these protein candidates, to elucidate how metal stress results in the loss-of-function of metalloproteins.

The structural and biochemical analysis of the zinc-de pendent regulator SczA, has revealed its importance in regulating zinc efflux through CzcD. This work has recently been accepted for publication in Molecular Microbiology.

Research Papers:

Publications
Hassan KA, Pederick VG, Elbourne LOH, Paulsen IT, Paton JC, McDevitt CA, Ei jkelkamp BA (2017). Zinc stress induces copper depletion in Acinetobacter baumannli, BMC Microbiology. Accepted article.
Eijkelkamp BA, Pederick VG, Plumptre CD, Harvey RM, Hughes CE, Paton JC and McDevitt, CA. The critical role of zinc in pneumococcal pathogenesis. Manuscript in Preparation.

Conference Presentations
McDevitt CA. The role of transition metal ions at the host-pathogen interface. The Australian Society for Microbiology, Annual Scientific Meeting, 2016, Perth, Australia
McDevitt CA. Prokaryotic chemical biology: new tools and methods. ABC Transporter Symposium, 2016, Canberra, Australia.
Eijkelkamp BA. The critical role of zinc in pneumococcal pathogenesis. UofA School of Biological Sciences Research Day, 2016, Adelaide, Australia.
Eijkelkamp BA. The critical role of zinc in pneumococcal pathogenesis. Adelaide Protein Group Early Career Awards, 2016, Adelaide, Australia. ECR Award Winner.

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