MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress

Abstract

Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

“…Earlier studies suggested that the BCAA biosynthetic pathway plays a role in pH homeostasis and IlvC has even been observed to be up-regulated at low pH (5.0) in Streptococcus mutans [20] and IlvC inactivation led to growth retardation at pH=5.5 [33]. Reduced survival of IlvA KD Mtb -Ra was also observed at lower pH (5.5 and 4.5) [5] as well as in IlvC KD of Mtb -Ra, as in the present study.…”

“…The cloning and expression studies were performed as per published protocols [5] and are detailed in Supplementary Data S1 and Fig. S4.…”

The MarR-Type Regulator MalR Is Involved in Stress-Responsive Cell Envelope Remodeling in Corynebacterium glutamicum

“…Among the genes showing a slightly increased mRNA level in response to malR expression, we also found the ilvA gene, encoding a threonine-dehydratase that is necessary for the production of isoleucine (Sharma et al, 2016). Isoleucine is a branched chain amino acid and, together with acetyl-CoA, an important precursor for the generation of branched chain fatty acids, which are part of the bacterial cell membrane.…”

Targeting amino acid metabolism of Mycobacterium tuberculosis for developing inhibitors to curtail its survival

“…It was observed that ilvA knockdown (KD) strain in H37Ra exhibited impaired growth on isoleucine depleted media and against oxidative stress induced by H 2 O 2 and NO. Further, the ilvA KD modified cell wall lipid composition and was vulnerable to antibiotics such as streptomycin, levofloxacin and rifampicin 56 . Isoleucine is the final product of threonine degradation pathway.…”