Transcriptional profiling of the clpX mutant in Bacillus anthracis reveals regulatory connection with the lrgAB operon

Abstract

ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne

“…0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; IBI Scientific, Peosta, IA, United States) was added when inducing the expression plasmid pUTE657. Construction of Streptococcus pyogenes Δ dltA , B. anthracis strain Δ clpX and wild-type clpX complementation plasmid (Δ clpX + p clpX ) along with the control strains of wild-type and Δ clpX containing the empty inducible plasmid pUTE657 were described previously ( Kristian et al, 2005 ; McGillivray et al, 2009 ; Claunch et al, 2018 ). All other strains were constructed in this study.…”

“…A substitution mutation at position 264 from isoleucine (ATT) to glutamic acid (GAA) of the B. anthracis clpX gene was made in the previously constructed clpX expression plasmid ( Claunch et al, 2018 ) to yield the clpX I 264 E plasmid. To do this, site-directed mutagenesis (NEB, Ipswich, MA, United States) was performed using the primers described in Table 1 following manufacturer’s instructions.…”

“…Bacillus anthracis , the etiological agent of the deadly disease Anthrax, is a gram-positive, spore forming bacterium. Previous work in our lab identified the clpX gene as essential for virulence in B. anthracis as well as resistance to antimicrobials that target the cell-envelope ( McGillivray et al, 2009 , 2012 ; Claunch et al, 2018 ). ClpX is a regulatory ATPase that functions in one of two ways, either as an independent chaperone or together with the caseinolytic peptidase (ClpP) to form the ClpXP protease ( Wawrzynow et al, 1995 ; Baker and Sauer, 2011 ).…”

The Potential of Human Peptide LL-37 as an Antimicrobial and Anti-Biofilm Agent

“…While metalloproteases assist in the pathogenesis of several bacteria, metalloprotease inhibitors can be effective in blocking the harmful effects on LL-37, such as the EDTA, 1,10-phenanthroline [29] and LasB inhibitors [72]. Other proteases implicated in LL-37 resistance include the ClpXP intracellular protease [74], the SpeB [32] and SspB secreted cysteine proteases [75], and the PASP [64], aureolysin and V8 secreted proteases [75].…”

“…In some cases, multiple genes have the same effect on a bacterium because the expression of one gene may be dependent on the expression or presence of another gene, or one gene may be a regulator of the target gene; therefore, a mutation in one gene has an effect on both genes. For example, dltC is regulated by CiaRH in Streptococcus mutans biofilm cells [37], the expression of lrgAB is dependent on the presence of ClpX [74], and zapB is needed for the recruitment and activity of EnvC [107]. In some cases, the precise effect of the gene is unknown, although there are suggested mechanisms based on the known role of the gene in the bacteria [30,64].…”