Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro

Abstract

Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

Figure 1

Clustal alignment of the USPs of M. tuberculosis. The 9 tandem domain proteins show a high degree of similarity across two key conserved motifs (DGS and G2×G9×GS, indicated above the sequence by short and long horizontal bars, respectively), but are less similar across the other regions of the proteins. Rv1636 is the only single domain USP present in Mtb. Rv2319c lacks some of the highly conserved residues present in all of the other USPs. Rv2005c, Rv2623, Rv2026c and Rv1996 in particular possess a high degree of similarity with each other in their second USP domain.

Figure 2

Genome context of the M. tuberculosis usp genes studied in this work.

Figure 3

Hypoxic survival of M. tuberculosis H37Rv usp mutants. The Mtb tandem domain usp mutants were cultured to hypoxic stationary phase and survival followed for 130 days. The viability (CFUs) was measured at several time-points to determine if the mutants possess any survival defect under these conditions. Error bars represent the standard deviations of 3 independent cultures.

Figure 4

The M. tuberculosis usp mutants exhibited no survival defect in response to a range of stress conditions. A number of other stress conditions were also tested (see Table 1), but no survival phenotype was observed when comparing the usp mutants to the wildtype H37Rv. As examples, the results for A) low pH and B) Nitrosative stress (5 mM GSNO) are shown. Error bars represent the standard deviations of 3 independent cultures.

Figure 5

hspX promoter activity in M. tuberculosis H37Rv and ΔRv2026c. The plasmid pMH108 carrying the hspX promoter region upstream of the firefly luciferase genes was transformed into Mtb H37Rv and ΔRv2026c. The promoter activity was measured in cultures grown under oxygen-sufficient conditions to mid-exponential phase and stationary phase and in cultures grown to hypoxic stationary phase. Relative light units (RLU) were determined and normalised using the OD of the cultures. The hspX promoter was seen to be active in both the wildtype and the ΔRv2026c mutants strain. Error bars represent the standard deviations of 3 independent cultures.

Figure 6

Intracellular survival of the usp mutants in the murine macrophage-like cell line J774 plus IFN-gamma. The usp mutants were tested for a survival defect in a macrophage cell line by determining CFU/ml over a 3-day time course. A similar curve was obtained for the inactivated macrophage infection.