Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses

Abstract

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.

Effects of Simulated Microgravity on the Physiology of Stenotrophomonas maltophilia and Multiomic Analysis

“…GO terms with a value of p < 0.05 were identified as significantly enriched by DEGs. R scrip was used to estimate the meaningful statistical enrichment of the DEGs in KEGG pathways (Wang et al, 2020).…”

Genome Sequence and Phenotypic Analysis of a Protein Lysis-Negative, Attenuated Anthrax Vaccine Strain

“…This resulted in negative proteolysis of PNO2D1 (Figure 3C). Furthermore, nprR knockout in A16Q1 [21] showed that extracellular protease activity was greatly reduced in the deletion mutant A16Q1ΔnprR (Figure 3C).…”

“…Nontoxigenic encapsulated strain A16Q1 (pXO1 − , pXO2 + ) was obtained from toxigenic encapsulated strain A16 using plasmid incompatibility [20]. A16Q1∆nprR was an nprR-deletion strain constructed as per Wang et al [21,22].…”

“…Hemolysis, phospholipase, and proteolysis of the strains were observed using specific plates. The extracellular protease activity phenotypes were investigated on milk agar plates [21,30]. Hemolysis of the B. anthracis strains was tested on Colombia blood agar [31].…”

Inhalable vaccine of bacterial culture supernatant extract mediates protection against fatal pulmonary anthrax

“…The B. anthracis V770-NP1-R strain used in AVA preparation is unencapsulated and has low protease activity [ 15 ]. Our previous work demonstrated that PA was upregulated and its degradation was inhibited in a mutant nprR -deficient B. anthracis strain [ 16 ]. Therefore, we hypothesized that a reduction in protease activity could mitigate the degradation of multiple known and unknown antigens secreted by the vaccine strain.…”