Characterization of mutants defective in D-Ala incorporation into lipoteichoic acid (LTA)

3.2.2.1. Construction and characterization of deletion mutants ΔdltB, ΔdltC, ΔvirR, ΔvirS and Δdal

It was of interest to study the interplay of the virR regulatory system and the dlt operon for survival and virulence of L. monocytogenes. Previous studies with a ΔdltA deletion mutant in L. monocytogenes exhibited increased susceptibility to cationic peptides (such as Nisin, Colistin and Polymyxin B) and severely impaired infection properties in a mouse model and in vitro with various cell lines (1). The authors concluded that D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L.

monocytogenes. A ΔvirR deletion mutant was shown to be more strongly impaired in virulence leading to decreased bacterial loads in infected mice and diminished entry of Caco2 cells. In this work, deletion mutants were constructed for genes of the dlt operon as well as virR and virS genes. Specifically, the dlt operon of L. monocytogenes was characterized by constructing D-Ala deficient mutants lacking dltB or dltC. Attempts to generate deletion strains for dltA and dltD were not successful even though ΔdltA mutants have been reported (1, 95).

In Listeria monocytogenes, the conversion of L-alanine to D-alanine is performed by the alanine racemase encoded by dal (153). The bacterium also has an alternative pathway to generate D-alanine through the activity of D-amino acid aminotransferase (dat).

Simultaneous mutation of the genes dal and dat leads to a dependence on exogenous alanine for proper growth (153). To understand the importance of the alanine racemase for D-alanine assembly into LTAs, a deletion mutant of the dal gene was also generated in this work.

The phenotypes of the ΔdltB, ΔdltC, ΔvirR, ΔvirS and Δdal mutants were examined with various assays for morphology, growth, autolysis and motility. None of the strains showed significant differences compared to the wild type L. monocytogenes EGDe (data not shown). However, considerable changes were found when the mutants were tested for antibiotic resistance. An antibiogram revealed increased sensitivity of the Δdal mutant to a few cephalosporins of different generations (Cefuroxim (2nd), Cefotaxim (3rd), Ceftriaxon (3rd) and Cefepim (4th)). A much stronger growth defect was seen with antibiotics that act as cationic peptides, such as Polymyxin B, Gallidermin and Colistin. The growth of deletion strains ΔdltB, ΔdltC, ΔvirR and ΔvirS was completely inhibited as shown in Figure 33A-C. In contrast, the Δdal strain had only a slight growth defect in the presence of cationic peptides.

The cationic peptides initially tested so far are of bacterial origin and have no relevance for L. monocytogenes during host infection. Therefore, we also tested a cationic peptide produced by eukaryotic host cells. The LL-37/human cationic antimicrobial protein 18 (LL37) consists of 37 amino acids and is synthesized by macrophages, neutrophiles, and epithelial cells (providing antimicrobial protection to skin and the lining of the urinary tract).

LL37 is a member of the cathelicidins and plays a major part in the innate immune response in the respiratory epithelium (156). Growth in the presence of LL37 (kindly provided by R.

Bals (Institute of Pneumology, Marburg University) was assessed for the ΔvirR and ΔvirS mutants. All strains reached stationary phase after almost 8 hours when cultured in BHI broth

(Fig. 33D). However, growth of the ΔvirR or ΔvirS deletion strains was completely arrested in broth supplemented with the antimicrobial peptide (Fig. 33D, white columns). Proliferation of the wild type EGDe, though not inhibited, was slightly delayed during cultivation with 50 μg/ml of the LL37 peptide.

Figure 33: Growth of L. monocytogenes EGDe wild type and of deletion mutants Δdal, ΔdltB, ΔdltC, ΔvirR and ΔvirS in the presence of cationic peptides. Bacteria were cultured in BHI broth supplemented with (A) 35 μg/ml Colistin, (B) 20 μg/ml Polymyxin B or (C) 0.5 μg/ml Gallidermin and the OD600 was measured hourly. The sensitivity to human CAMP LL37/

/hCAP-18 was tested for EGDe wild type and ΔvirR and ΔvirS deletion mutants (D). Growth of L. monocytogenes EGDe in BHI supplemented with water (black columns) or 50 μg/ml LL37 /hCAP-18 (white columns) was monitored by measuring the optical density at 600 nm.

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The importance of the dlt-operon and the VirR/S system for bacterial resistance against CAMPs was also analyzed by quantitative real time-PCR. The EGDe wild type was cultured in BHI supplemented with Polymyxin B or Colistin. Cells were harvested at exponential phase and RNA was isolated and reverse transcribed. The expression pattern of dltA, mprF, virR and virS was tested by quantitative real time PCR and compared to that of bacteria cultured in BHI only. Whereas the dltA and mprF genes were found to be

up-regulated during growth in the presence of Colistin or Polymyxin B (Fig. 34), genes of the VirR/VirS regulon showed no significant changes.

Figure 34: Quantitative real-time PCR of dltA, mprF, virR and virS inEGDe in the presence of cationic peptides. L.

monocytogenes EGDe was grown fo exponential phase in BHI supplemented with (A) 40 μg/ml Colistin or (B) 30 μg/ml Polymyxin B and cells were harvested for RNA isolation and subsequent RT real time PCR. The genes dltA, mprF, virS and virR were quantified according to Chatterjee (2006) (28) by using standard curves obtained from genomic DNA. The values were normalized to bacteria grown in BHI without addition of antibiotics.* p < 0.05.

Bacterial sensitivity to ethanol stress was also tested. Whereas the deletion mutants for virR, virS, dltB and dltC exhibited growth similar to the EGDe wild type, the dal deletion mutant was shown to be more vulernable against ethanol (Fig. 35).

Figure 35: Sensitivity of deletion mutants Δdal, ΔdltB, ΔdltC, ΔvirS and ΔvirR to ethanol stress.

A B

3.2.2.2. Virulence properties of the mutant strains

We were also interested in the virulence properties of the newly created deletion strains of L. monocytogenes. Invasion was tested as previously described with human epithelial cells. Surprisingly, the deletion strains displayed a large difference in their invasion of Caco2 versus Hela cells. Whereas the invasion rate of Hela cells was almost wild type level (with the exception of ΔdltB with 38 %), there was a strong invasive defect with the Caco2 cell line (Fig. 36A). The Δdlt and Δvir deletion mutants had invasion rates of only 0.4 to 3%.

Figure 36: In vitro virulence assays with L. monocytogenes EGDe and deletion mutants ΔdltC, ΔdltB, ΔvirR, ΔvirS and Δdal.

(A) Invasion into the human epithelial cell lines Hela and Caco2, (B) intracellular replication in P388D1 macrophages and (C) intracellular spreading in L-929 fibroblasts.

Our next approach to analyze the virulence of the CAMP sensitive strains was to test intracellular survival in the macrophage cell line P388D1. Intracellular replication was found to be dramatically decreased with all deletion strains (Fig. 36B). The ability of the deletion strains to spread from one cell to another was assayed by infection of fibroblast monolayers

and monitoring plaque formation after incubation for several days. All mutant strains were capable of successfully infecting the L-929 cell line however plaque sizes of the ΔdltB/C and ΔvirR/S strains were about 50% reduced compared to the wild type (Fig. 36C). Significant inabilit of the ΔvirR and ΔvirS deletion mutants to properly infect Caco2 cells raised the question of whether the expression of listerial internalin A is reduced. This protein has been shown to be critical for cellular adhesion and internalization into Caco2 cells by binding to the glycoprotein E-Cadherin (102). We therefore verified expression of InlA and InlB expression in both the ΔvirR and ΔvirS mutant by performing immunoblot analysis. Despite the strong variation of infection into Hela versus Caco2 cells, there were no significant differences detectable in internalin levels using monoclonal antibodies (data not shown). To determine the infection step that is inhibited in the ΔvirR and ΔvirS strains, bacteria were stained using an ActA-directed monoclonal antibody during in vitro infection of the epithelial cell line PTK2. By means of immunofluorescence microscopy we found fewer bacteria in the host cells but no apparent defect in adhesion or phagosomal escape during infection.

3.2.2.3. Role of the D-alanylation of teichoic acids for immune activation

In S. aureus the substitution of D-alanine on teichoic acid has been shown to also be important for cytokine induction (107). In Lactobacillus plantarum the deletion of dlt significantly reduces cytokine release in a Toll-like receptor 2 (TLR2)-dependent manner.

Nevertheless, studies performed in our lab with ΔdltB and ΔvirS mutants of L. monocyto-genes could not confirm this effect in Hela cells transfected with TLR2 (data not shown).

There were no significant differences in TLR2-dependent NF-κB activation induced by L.

monocytogenes wild type versus the deletion strains.