MnmE, a Central tRNA-Modifying GTPase, Is Essential for the Growth, Pathogenicity, and Arginine Metabolism of Streptococcus suis Serotype 2

Abstract

Streptococcus suis is an important pathogen in pigs and can also cause severe infections in humans. However, little is known about proteins associated with cell growth and pathogenicity of S. suis. In this study, a guanosine triphosphatase (GTPase) MnmE homolog was identified in a Chinese isolate (SC19) that drives a tRNA modification reaction. A mnmE deletion strain (ΔmnmE) and a complementation strain (CΔmnmE) were constructed to systematically decode the characteristics and functions of MnmE both in vitro and in vivo studies via proteomic analysis. Phenotypic analysis revealed that the ΔmnmE strain displayed deficient growth, attenuated pathogenicity, and perturbation of the arginine metabolic pathway mediated by the arginine deiminase system (ADS). Consistently, tandem mass tag -based quantitative proteomics analysis confirmed that 365 proteins were differentially expressed (174 up- and 191 down-regulated) between strains ΔmnmE and SC19. Many proteins associated with DNA replication, cell division, and virulence were down-regulated. Particularly, the core enzymes of the ADS were significantly down-regulated in strain ΔmnmE. These data also provide putative molecular mechanisms for MnmE in cell growth and survival in an acidic environment. Therefore, we propose that MnmE, by its function as a central tRNA-modifying GTPase, is essential for cell growth, pathogenicity, as well as arginine metabolism of S. suis.

Keywords: MnmE; Streptococcus suis (S. suis); arginine deiminase system; growth; pathogenicity; tRNA modifying guanosine triphosphatase; tandem mass tag-based quantitative proteomics.

Figure 1

Confirmation and Characterization of the isogenic mutant ΔmnmE. (A) Combined PCR analyses of the ΔmnmE mutant. Lanes 1 to 3 represent the amplification of the upstream gene, mnmE gene, and downstream gene of SC19 using the primer set mnmE-F/mnmE-R, 1453-F/1453-R, and 1455-F/1455-R. Lanes 4 to 6 represent the amplification of the upstream gene, mnmE gene and downstream gene of ΔmnmE using the primer set mnmE-F/mnmE-R, 1453-F/1453-R, and 1455-F/1455-R. Lanes 7 to 9 represent the amplification of the upstream gene, mnmE gene and downstream gene of CΔmnmE using the primer set mnmE-F/mnmE-R, 1453-F/1453-R, and 1455-F/1455-R. (B) Confirmation of the ΔmnmE mutant by RT-PCR. Lanes 1, 4, and 7 represent the amplification of upstream gene of mnmE using the primer set 1453-F/1453-R. Lanes 2, 5, and 8 represent the amplification of mnmE using primer set mnmE-F/mnmE-R. Lanes 3, 6, and 9 represent the amplification of downstream gene of mnmE using the primer set 1455-F/1455-R. Lanes 1, 2, and 3 use cDNA of SC19 as templates, whereas Lanes 4, 5, and 6 use cDNA of ΔmnmE as templates, Lanes 7, 8, and 9 use cDNA of CΔmnmE as templates. (C) Growth curves of the strains. Bacterial cell density was measured spectrometrically at 600 nm. Data were collected at the indicated times. (D) CFU count of the strains. Separate aliquots of the bacterial suspensions were serially diluted and plated to determine CFU numbers per milliliter. Data were collected at the indicated times.

Figure 2

MnmE contributes to SS2 pathogenicity in vivo and in vitro. (A) Survival curves for mice in experiment infection. Ten mice in each group were separately injected intraperitoneally with 3 × 10⁹ CFU/mice of SC19 and ΔmnmE. Ten mice were inoculated with saline and served as negative control. Significant difference in survival between different groups were analyzed by Log Rank test (p < 0.01). (B) Cell-associated bacteria recovered after incubation with HEp-2 cells. The mutant strain ΔmnmE showed significantly reduced levels of adherence to HEp-2 cells compared with the degree of adherence of SC19 and CΔmnmE (p < 0.05). (C) Bacteria invasion of HEp-2 cells. The mutant strain ΔmnmE showed significantly reduced levels of invasion of HEp-2 cells compared with that of SC19 and CΔmnmE (p < 0.01). Statistical significance was determined by two-tailed t-test (^*p < 0.05; ^**p < 0.01).

Figure 3

Bacteria loads in different mouse organs. Bacteria loads in (A) brain, (B) lung, (C) in spleen, and (D) in blood. Mice were inoculated intraperitoneally with 1 × 10⁸ CFU of a 1:1 mixture of mid-log phase SC19 and ΔmnmE. The survival strains were enumerated by plating serial dilutions of the samples on selective plates. Data are the result of CFU/g or CFU/ml in different organs analyzed per sample ± SEM. Statistical significance was determined by two-tailed t-test (ns, p > 0.05; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 4

Regulation of the ADS by MnmE. (A) AD activities of different S. suis. Results were expressed as nanomoles of citrulline produced per hour per milligram of whole cell protein. (B) Production of ammonia in different S. suis. Ammonia production in supernatant of different S. suis is given as μg ammonia / ml. Data are presented as the mean ± SEM of a representative experiment performed intriplicate. Statistical significance was determined by two-tailed t-test (^**p < 0.01; ^***p < 0.001). (C) Schematic representation of S. suis arginine metabolic pathways. The core ADS enzymes (ArcA, ArcB, ArcC) facilitating the conversion from arginine to ornithine are depicted in orange. Metabolic intermediates are indicated in blue. The input of energy in terms of phosphate or phosphate derivatives (ADP) are marked in red, non-catabolized, and excreted products have a green color.

Figure 5

Classification of differentially expressed proteins in S. suis according to GO annotation.