Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection.

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for 'thermal restriction'. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.

Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production.

The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. KEY POINTS: • Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production • Triple deletion mutant with transketolase overexpression increased succinate yield by 180% • P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization.