Activity of Cathelicidin Peptides against Simkania negevensis

The in vitro activity of six cathelicidin peptides against the reference strain Z of Simkania negevensis was investigated. Five peptides—PG-1, Bac7, SMAP-29, BMAP-27, and BMAP-28—proved to be active at very low concentrations (1 to 0.1 μg/mL), while LL-37 cathelicidin was ineffective even at a concentration of 100 μg/mL. In comparison to chlamydiae, S. negevensis proved to be more susceptible to the antimicrobial peptides tested.


Introduction
Simkania negevensis is an obligate intracellular Gram-negative bacterium belonging to the family of Simkaniaceae in the order Chlamydiales [1], discovered as a contaminant in a variety of cell cultures [2], and is able to grow also in various environmental free-living amoebae such as Acanthamoeba poliphaga [3].
Epidemiologic studies have reported a human widespread exposure to this bacterium [4,5], both in healthy subjects and in association with respiratory diseases in infants and adults [6][7][8][9][10]. Coinfections with other pathogens have been described, such as respiratory syncytial virus in children and influenza virus and other bacterial species in adults [7,8,11]. A possible association between S. negevensis and acute rejection in lung transplant recipients has been suggested [12]. S. negevensis DNA has also been amplified from an aortic aneurysm [13,14].
Several studies [17][18][19][20] demonstrated the in vitro antichlamydial activity of peptides, such as cathelicidins, stored by mammalian leucocytes and show an antimicrobial activity against bacteria, fungi, protozoa, and enveloped viruses. With regard to the mode of action of these peptides against chlamydiae, previous investigations on the activity of protegrins [21] suggested an initial binding to lipopolysaccharide followed by insertion into the membrane that results in permeabilization. Yasin et al. [22] demonstrated that the antichlamydial activity of protegrins resided especially in residues 5 to 15 of native protegrin-1 and required both intramolecular disulfide bonds.
In the present study, we investigate the in vitro activity of six antimicrobial peptides against the reference strain Z of S. negevensis.

Materials and Methods
The cathelicidin peptides-PG-1 from pig, Bac7 (1-35) from cattle, SMAP-29 from sheep, LL-37 from human, BMAP-27, and BMAP-28 from cattle-were chemically synthesized, purified, characterized, and provided as lyophilized peptide, as previously reported [19]. The reference strain Z of S. negevensis (American Type Culture Collection VR-1471) was grown in LLC-MK2 cells [23] in 6-large well plates and elementary bodies (EBs) were purified by use of sucrose gradients [24], resuspended in sucrose-phosphate-glutamic Stock solutions of each peptide at 1 g/liter were prepared in phosphate-buffered saline (PBS), pH 7.4, and stored frozen in 20-µL aliquots at −80 • C until used. To determine the lowest peptide concentration required to achieve ≥50% reduction of inclusions with respect to untreated control, the various peptides were diluted tenfold from 200 to 0.02 µg/mL, in a volume of 100 µL with PBS in polypropylene tubes. An equal volume of 5 × 10 5 inclusion-forming units (IFU)/mL of purified S. negevensis EBs in SPG medium was then added, so obtaining a final cathelicidin concentration ranging from 100 to 0.01 µg/mL in a mixture of PBS and SPG at a final pH 7.4. EBs untreated with cathelicidins were used as a control. After incubation at room temperature for 2 h, a 200 µL aliquot was added to 800 µL chlamydial growth medium and inoculated onto LLC-MK2 cells grown in 24well plates in a 5% CO 2 atmosphere. After centrifugation at 800 × g for 1 h at 33 • C and incubation at 35 • C for 72 h, the cultures were fixed and stained for the presence of inclusions by immunofluorescence using an in-house rabbit polyclonal hyperimmune serum raised against S. negevensis. The number of IFU per coverslip was counted in 40 microscopic fields using a ZEISS UV microscope at a magnification of ×200.
All tests were performed in triplicate and in three independent experiments.

Results and Discussion
The activities of the six cathelicidin peptides against S. negevensis are reported in Figure 1 and are the means of results from the three experiments. In comparison with the infectivity of the untreated control (mean value 74 IFU/mL), Bac7 and SMAP-29 reduced by >50% (<37 IFU/mL) the inclusion numbers of S. negevensis at a concentration of 0.1 µg/mL, whereas PG-1, BMAP-27 and BMAP-28 showed the same activity at a concentration of 1 µg/mL. All these five cathelicidins demonstrated a very slow decrease in their inhibitory effect while their concentration has been reducing from 100 to 1 or 0.1 µg/mL. LL-37 peptide did not exert any inhibitory effect, even at a concentration of 100 µg/mL.
In our previous study [19], the activity of six cathelicidins-PG-1, Bac7, SMAP-29, BMAP-27, LL-37 and BMAP-28-tested on chlamydiae of human and animal origin showed that five of them had inhibitory effect on Chlamydia trachomatis strains, SMAP-29 being the most active peptide at a concentration of 10 µg/mL; on the contrary, LL-37 peptide was ineffective even at a concentration of 80 µg/mL. With regard to the other chlamydial species tested, only Chlamydophila pneumoniae and Chlamydophila felis were sensitive to 10 µg/mL of SMAP-29 and 80 µg/mL of SMAP-29/Bac7, respectively.
In the present investigation, the same six cathelicidins were tested against the reference strain Z of S. negevensis. In comparison with chlamydiae, S. negevensis was not sensitive to LL-37 even at the concentration of 80 µg/mL, but very sensitive to all the other five cathelicidins that showed an inhibitory effect at very low concentrations from 1 to 0.1 µg/mL. To our knowledge no study on S. negevensis susceptibility to cathelicidins has been performed, until now. These preliminary results suggest a higher sensitivity of S. negevensis to cathelicidins respect to chlamydiae. A previous study showed differences in the susceptibility of different C. trachomatis serovars to antimicrobial peptides, suggesting that limited variations in the disulfide bonding in the membrane structure of these organisms may influence their susceptibility to peptides [18]. The membrane structure of S. negevensis probably differs significantly from that of other members of Chlamydiales, as monoclonal antibodies recognizing family-specific epitopes of chlamydial OMP-2, MOMP, and LPS did not bind S. negevensis antigens [2,25]; in addition, only a relatively small number of common epitopes were shown to be shared when S. negevensis and other Chlamydiales members were tested for cross-reactivity by Western blot using polyclonal antisera [26]. The higher International Journal of Peptides 3 susceptibility of S. negevensis to cathelicidins in comparison to chlamydiae could be referred, to a great extent, to its different polypeptides pattern.