The dltABCD Operon of Bacillus anthracis Sterne Is Required for Virulence and Resistance to Peptide, Enzymatic, and Cellular Mediators of Innate Immunity

Abstract

In the environment, the gram-positive bacterium Bacillus anthracis persists as a metabolically dormant endospore. Upon inoculation into the host the endospores germinate and outgrow into vegetative bacilli able to cause disease. The dramatic morphogenic changes to the bacterium during germination and outgrowth are numerous and include major rearrangement of and modifications to the bacterial surface. Such modifications occur during a time in the B. anthracis infectious cycle when the bacterium must guard against a multitude of innate immune mediators. The dltABCD locus of B. anthracis encodes a cell wall D-alanine esterification system that is initiated by transcriptional activation during endospore outgrowth. The level of transcription from the dltABCD operon determined B. anthracis resistance to cationic antibacterial peptides during vegetative growth and cationic peptide, enzymatic, and cellular mediators of innate immunity during outgrowth. Mutation of dltABCD was also attenuating in a mouse model of infection. We propose that the dltABCD locus is important for protection of endosporeforming bacteria from environmental assault during outgrowth and that such protection may be critical during the establishment phase of anthrax.

Transcriptional Profiling of Bacillus anthracis during Infection of Host Macrophages

“…Figure 3 shows survival curves for mice that were inoculated via intratracheal injection with 1.5 ϫ 10 4 spores of either the parent Sterne 34F 2 or mutant ⌬GBAA1941 strain. This dose of Sterne 34F 2 spores represents roughly a 90% lethal dose in this model system (21), and only 2 of the original 27 mice in the parent group survived the infection. In contrast, nearly one-half (12 of 26) of the mice inoculated with the mutant strain survived the infection.…”

“…Mouse infection was done essentially as described previously (21). Briefly, DBA/2 mice were anesthetized by intraperitoneal injection of ketamine (2.5 mg/mouse) and xylazine (0.1 mg/mouse), and a small incision was made through the skin over the trachea.…”

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

“…However, in the spore-forming bacterium B. anthracis, the dlt genes are turned on during spore germination in the host and are necessary for resistance to innate host defences (Fisher et al, 2006). Because a potential regulatory protein is located immediately adjacent to the dlt operon, we examined the regulation of its expression.…”

“…Results were calculated using the comparative cycle threshold method (Schmittgen & Livak, 2008), in which the amount of target mRNA is normalized to that of an internal control transcript (rpoC). The two-tailed Student's t test was used to analyse the data.Quantification of D-alanine ester content in teichoic acids.Analysis of ester-linked D-alanine incorporation into cell walls was performed as described elsewhere, with minor modifications (Hyyrylainen et al, 2000;Fisher et al, 2006). Cultures (50 ml) of wild-type (JIR8094), dltD mutant (MC120) and the complemented strain (MC129) were grown to OD 600 0.2-0.25 in BHIS anaerobically at 37 uC and harvested by centrifugation, and the cell pellets were stored at 280 uC prior to analysis.…”

“…All four proteins are required for successful addition of D-alanine to the cell wall (Neuhaus & Baddiley, 2003). Incorporation of D-alanine into teichoic acid has been demonstrated to increase resistance of bacteria to CAMPs and bacteriolytic enzymes, as well as contribute to the virulence of pathogens such as Bacillus anthracis (Fisher et al, 2006), Listeria monocytogenes (Abachin et al, 2002), Streptococcus pneumoniae (Wartha et al, 2007) and Staphylococcus aureus (Weidenmaier et al, 2005).…”