There is a significant amount of knowledge regarding the competition for iron between bacteria and their hosts (as reviewed in reference 20), but less attention has been paid to the mechanisms that limit zinc for microbes growing in association with hosts. Competition for zinc is suggested by the fact that several bacterial species require a functional high-affinity ZnuABC transport system for colonization and/or virulence, including C. jejuni
, E. coli
, Salmonella enterica
, Proteus mirabilis
, Haemophilus influenzae
, and Brucella abortus
). The presence of the microbiota affects metabolic functions and subsequent zinc utilization, of which little is known. Beyond uncovering basic new knowledge about an important biological process, the value of understanding the role of ZnuABC function in the interaction of pathogenic bacteria and their hosts is enhanced by the fact that a Salmonella znuABC
mutant strain provides protection against lethal challenge in a mouse model (24
). The ZnuABC transporter is required for Salmonella
persistence during an inflammatory response due to zinc limitation resulting from expression of the metal-binding antimicrobial protein calprotectin by neutrophils. znuA+ Salmonella
had a significant growth advantage over ΔznuA Salmonella
in the inflamed intestines of colonized mice. When infection was established without inflammation or in mice lacking the genes for calprotectin, the growth advantage of the ZnuA-expressing Salmonella
was lost (25
). Growth limitation of incoming microbes by limiting essential elements, such as zinc or iron, has been termed “nutritional immunity” (26
). Inflammation is not a major outcome from infection of chickens by C. jejuni
(27; L. M. Gielda, N. Iovine, K. Eaton, and V. DiRita, unpublished data), but our work demonstrates that the microbiota can also contribute to limiting the bioavailability of micronutrients. These studies highlight the importance of zinc acquisition for both pathogenic and commensal organisms in the intestines and demonstrate how basic metabolic activities of pathogens are as critical to their association with hosts as are frank virulence traits, such as adhesins, toxins, and invasion systems.
Lower quantities of trypsin, aminopeptidase Ey, and procarboxypeptidase A in limited-microbiota chicks suggest that there are metabolic changes dependent on the presence of a microbiota (). This alteration in host metabolism may explain the higher level of zinc in limited-microbiota cecal contents, as several of the digestive proteins identified in the conventional cecal contents bind zinc. The decrease in zinc-binding proteins, or a decreased rate of metabolism or cell proliferation in the intestines (functions that require the use of zinc), would cause an increase in available zinc. However, the effects of the microbiota on digestive functions of the host are poorly understood.
Intestinal bacteria have been implicated in the degradation of secreted digestive enzymes, including trypsin, chymotrypsin, glycoside hydrolases, and aminopeptidase N (28-30
). In a recent study, conventionalization of neonatal gnotobiotic pigs resulted in higher aminopeptidase N expression than in germfree pigs (30
). However, enzymatic activity was reduced in conventionalized and E. coli
-infected pigs, suggesting protein destabilization by the resident bacteria, which was supported by E. coli
degradation of purified aminopeptidase in vitro
). Our work demonstrated an abundance of aminopeptidase Ey in conventional chicks compared to that in limited-flora chicks, correlating with the increase of aminopeptidase N expression in the aforementioned study (30
). Although deactivation of the protein was not observed in conventionalized chicks, differences between the microbiota compositions of chicks and pigs may explain the observation, as Lactobacillus fermentum
and Klebsiella pneumoniae
do not deactivate aminopeptidase N activity as well as E. coli
). These studies demonstrate that the host metabolism may be altered based on the bacterial enzymatic activities and composition of the residing microbiota.
The presence of ovotransferrin in the chicken cecal contents, a protein found in high abundance in chicken egg whites capable of binding ions such as iron and zinc may deprive C. jejuni
of zinc necessary for growth. Ovotransferrin has been shown to have antibacterial effects against Pseudomonas
, E. coli
, Streptococcus mutans
, Staphylococcus aureus
, Bacillus cereus
, and Salmonella enterica
serovar Enteritidis (31-33
). Research into whether growth inhibition of the ΔznuA C. jejuni
mutant in chick cecal contents is due to the presence of a specific microbiota-dependent zinc-binding protein, competition with the dense microbiota population, or alterations in the host immune or metabolic functions is under way.
We have demonstrated that quantities of zinc in the gastrointestinal tract are reduced in conventional chicks compared to limited-flora chicks, suggesting that the microbiota affect the availability of this trace element. Without its high-affinity zinc transporter, C. jejuni is unable to compete in conventional chicks. Further research is needed to understand how the microbiota-dependent metabolic changes of the host affect the availability of zinc and other micronutrients for commensal organisms and for incoming pathogens as well.