Analysis of the Isoprenoid Biosynthesis Pathways in Listeria monocytogenes Reveals a Role for the Alternative 2-C-Methyl- d -Erythritol 4-Phosphate Pathway in Murine Infection

Abstract

Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce V␥9V␦2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes

HMBPP-deficientListeriamutant immunization alters pulmonary/systemic responses, effector functions, and memory polarization of Vγ2Vδ2 T cells

“…We then sought to determine whether removing gcpE from our attenuated Listeria strain would further impact bacterial replication and immunogenicity. This question needs to be addressed, as a study demonstrates that the HMBPP/MEP pathway influences virulence for a WT Listeria strain [31]. We confirmed that gcpE deletion in a WT strain of Listeria (10403S) resulted in decreases in bacterial loads in spleen and liver following i.v.…”

“…Our study represents the first in vivo comparative investigation dissecting importance of HMBPP/IPP coproduction versus IPP production for eliciting V␥2V␦2 T cell expansion, differentiation, and memory formation during intracellular bacterial immunization. A number of pathogens, including most detrimental ones that cause TB, cholera, diphtheria, and plague, indeed carry the MEP pathway for HMBPP/IPP coproduction without an additional mevalonate IPP pathway [3,31] and activate/expand V␥2V␦2 T cells. Our primate model and listerial mutant system provide a unique opportunity to document whether and how HMBPP deficiency during immunization with HMBPP-producing bacteria impacts in vivo responses of V␥2V␦2 T cells.…”

“…Whereas most bacterial pathogens capable of expanding V␥2V␦2 T cells only carry the MEP pathway for HMBPP/IPP coproduction without coexistence of the mevalonate pathway for IPP production, a deletion of a key gcpE gene in the MEP pathway would shut down HMBPP/IPP coproduction (HMBPP metabolizes further to IPP; ref. [30]) and render these pathogens nonviable or nonreplicating [31,32]. As Lm is the pathogen carrying MEP and mevalonate pathways for critical bacterial metabolism, we made use of our experience in genetic modification of attenuated Lm vaccine strain (Listeria ⌬actA prfA*) for immunization of macaques [14,27,28].…”

“…Previous studies have demonstrated that respiratory infections or immunization of macaques with HMBPP/IPP-coproducing bacteria, including Mtb and Yersinia pestis, can induce expansion or accumulation of V␥2V␦2 T cells in lungs, which correlates with improved clinical outcomes after pulmonary infections [26]. As these organisms only have the MEP pathway of isoprenoid biosynthesis, knockout of gcpE gene in the MEP pathway from these pathogens would eliminate HMBPP/IPP coproduction and thus, lead to a loss of the ability for these organisms to metabolize and survive [31]. The ability of the IPPproducing ⌬gcpE mutant to survive and replicate in an absence of the MEP pathway gave us a unique opportunity to dissect HMBPP production versus IPP production for expansion and differentiation of V␥2V␦2 T cells during infection or immunization with HMBPP/IPP-coproducing pathogens or vaccine vectors.…”

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

“…The importance of [4Fe-4S] cluster biosynthesis for these two enzymes was underlined by the recent demonstration that E. coli can survive a knockout of both Fe-S assembly systems, ISC and SUF, as long as mevalonate and the lower genes of the mevalonate pathway are provided to ensure a supply of IPP and DMAPP (Tanaka et al, 2016). It’s also interesting to note that while pathogenic strains of Listeria monocytogenes maintain a full DXP pathway as well as a mevalonate pathway, its non-pathogenic relative Listeria innocua has specifically lost the genes encoding IspG and IspH, suggesting that these two [4Fe-4S] enzymes may constitute a high metabolic or physiological burden (Begley et al, 2008). …”