Esx Paralogs Are Functionally Equivalent to ESX-1 Proteins but Are Dispensable for Virulence in Mycobacterium marinum

Abstract

Mycobacterium marinum is a nontuberculous pathogen of poikilothermic fish and an opportunistic human pathogen. Like tuberculous mycobacteria, the M. marinum M strain requires the ESX-1 (ESAT-6 system 1) secretion system for virulence in host cells. EsxB and EsxA, two major virulence factors exported by the ESX-1 system, are encoded by the esxBA genes within the ESX-1 locus. Deletion of the esxBA genes abrogates ESX-1 export and attenuates M. marinum in ex vivo and in vivo models of infection. Interestingly, there are several duplications of the esxB and esxA genes (esxB_1, esxB_2, esxA_1, esxA_2, and esxA_3) in the M. marinum M genome located outside the ESX-1 locus. We sought to understand if this region, known as ESX-6, contributes to ESX-1-mediated virulence. We found that deletion of the esxB_1 gene alone or the entire ESX-6 locus did not impact ESX-1 export or function, supporting the idea that the esxBA genes present at the ESX-1 locus are the primary contributors to ESX-1-mediated virulence. Nevertheless, overexpression of the esxB_1 locus complemented ESX-1 function in the ΔesxBA strain, signifying that the two loci are functionally equivalent. Our findings raise questions about why duplicate versions of the esxBA genes are maintained in the M. marinum M genome and how these proteins, which are functionally equivalent to virulence factors, contribute to mycobacterial biology.IMPORTANCEMycobacterium tuberculosis is the causative agent of the human disease tuberculosis (TB). There are 10.4 million cases and 1.7 million TB-associated deaths annually, making TB a leading cause of death globally. Nontuberculous mycobacteria (NTM) cause chronic human infections that are acquired from the environment. Despite differences in disease etiology, both tuberculous and NTM pathogens use the ESX-1 secretion system to cause disease. The nontubercular mycobacterial species, Mycobacterium marinum, has additional copies of specific ESX-1 genes. Our findings demonstrate that the duplicated genes do not contribute to virulence but can substitute for virulence factors in M. marinum These findings suggest that the duplicated genes may play a specific role in NTM biology.

Keywords: CFP-10; ESX-1; ESX-6; duplication; gene duplication; mycobacteria; type VII secretion.

FIG 1

M. marinum ESX-6 genes share similarities with ESX-1 genes. Comparison between paralogous genes at the ESX-1 locus and extended ESX-6 locus. The percent identity at the protein level is shown. For simplicity, the entire ESX-1 locus is not depicted; only genes with paralogs in the extended ESX-6 region are shown. The ESX-6 region is bracketed.

FIG 2

M. marinum ESX-6 genes share similarities with ESX-1 genes. (A) Alignment of the esxB and esxB_1 genes from M. marinum M. The translational start site is indicated by an asterisk. The arrows indicate the binding sites for ors148 and ors149 primers, which were used for qRT-PCR analysis of the esxB and esxB_1 genes. The alignment was performed using Clustal Omega and visualized using BoxShade. (B) Expression levels of esxB, esxB_1, and esxA were determined using qualitative RT-PCR. RT, reverse transcriptase. The groEL2 gene transcript was a control for cDNA quality. The no-RT control was a control for contaminating genomic DNA (gDNA). The figure is representative of three independent biological replicates. (C) Expression levels of esxB, esxB_1, and esxA determined using quantitative RT-PCR. esxB, esxB_1, and esxA transcripts were normalized relative to the level of the sigA gene. Error bars represent standard deviations of four technical replicates performed on two biological replicates (n = 8). Significance relative to the WT strain was determined using a one-way ANOVA (P < 0.0001) followed by a Dunnett's multiple-comparison test. (****, P ≤ 0.0001).

FIG 3

The esxB_1 gene and the extended ESX-6 region are dispensable for ESX-1 secretion and activity. (A) ESX-1 secretion assay. The cytosolic protein RNA polymerase subunit beta (RNAP-β) was a lysis control for supernatant fractions. Mpt-32, a Sec-secreted protein, served as a loading control. P, pellet, S, supernatant; comp, the ΔesxB_1/p_MopsesxB_1-esxA_3 complementation strain (Mops is the mycobacterial optimal promoter). (B) Sheep RBC hemolysis assay. The data represent at least four biological replicates, each with three technical replicates. The data were averaged, and the propagated error was calculated from the standard deviation from each replicate. The error bars represent the propagated error. Significance was calculated using an ordinary one-way ANOVA (P < 0.0001) followed by a Sidak's multiple-comparison test between all possible pairs. Significance relative to results with the WT are indicated (****, P < 0.0001; *, P = 0.0269). Note that there was no significant difference between results for the esxB_1 and esxB_1 and complemented strains. (C and D) ESX-1 secretion assay and sRBC hemolysis assay. The ΔextESX-6 strain includes deletion of the MMAR_0184-esxA_2 genes. The data from at least four biological replicates were averaged, and the propagated error was calculated from the standard deviation from each replicate. The error bars represent the propagated error. Significance was calculated using an ordinary one-way ANOVA (P < 0.0001) followed by Tukey's multiple-comparison test between all possible pairs. Significance relative to results with the WT strain is shown (****, P = 0.0001). (E) Infection of A. castellanii with M. marinum. Each infection was performed in technical duplicate on three biological replicates. Representative images are shown above. All images in the figure were adjusted with +40% brightness to aid in visualization. The ethidium homodimer-1-stained amoebae were quantified using ImageJ. All fields from three infections were averaged. The error bars represent the standard deviation between fields (n = 20). Significance was determined using an ordinary one-way ANOVA (P < 0.0001) followed by Dunnett's multiple-comparison test relative to results with the WT strain (****, P = 0.0001).

FIG 4

EsxB_1 and EsxA_1 can by secreted by Esx-1 and are functionally redundant. (A) ESX-1 secretion assay. Controls for the secretion assay are the same as those described in the legend to Fig. 2. In this assay, the EsxB and EsxA antibodies are recognizing EsxB_1 and EsxA_1/EsxA_3 for lanes 5 to 8. Data are representative of at least three biological replicates. (B) sRBC hemolysis assay. The data represent at least four biological replicates, each with three technical replicates. The data were averaged, and the propagated error was calculated from the standard deviation from each replicate. The error bars represent the propagated error. Significance was calculated using an ordinary one-way ANOVA (P < 0.0001) followed by Dunnett's multiple comparison for results compared to those with the WT strain (****, P < 0.0001). (C) qRT-PCR analysis measuring IFN-β expression normalized to actin expression at 24 h postinfection with M. marinum. The data are the average of four biological replicates, each with two technical replicates. The error bars represent the propagated error (n = 8 total). Statistical significance was determined using an ordinary one-way ANOVA (P < 0.0001) followed by Dunnett's multiple-comparison test comparing results with infection by the WT strain. (****, P = 0.0001; ***, P = 0.0003 and 0.0002 for pesxBA and pesxB_1-esxA_3, respectively).