Antimicrobial Activity of Inducible Human b Defensin-2 AgainstMycoplasma pneumoniae

Koichi Kuwano, Noriko Tanaka, Takashi Shimizu, Yutaka Kida

Department of Bacteriology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan

Received: 20 July 2005 / Accepted: 31 January 2006

Abstract. Defensins in innate immunity are known to play critical roles to protect the host frominfection by invasive microbes, including Gram-positive and -negative bacteria. However, little isknown about the interactions between defensins and mycoplasmas. Human b defensin (hBD)-2 andhBD-3, but not hBD-1, were found to exert strikingly antimicrobial activity against Mycoplasmapneumoniae. To elucidate the role of defensins in M. pneumoniae infection, a human pulmonarysquamous cell line EBC-1 was stimulated with M. pneumoniae and interleukin (IL)-1b. hBD-2 wasmarkedly upregulated by IL-1b as well as M. pneumoniae, but neither hBD-1 nor hBD-3 was apparentlyupregulated. Thus, the results suggest that inducible hBD-2 would play a critical role in the protection ofM. pneumoniae infection.

Antimicrobial peptides in innate immunity are widelyknown to play critical roles to protect the host frominfection by a variety of invasive microbes [8, 24]. Inmany vertebrates, the primary family of antimicrobialpeptides is the defensin, produced in neutrophils andepithelial cells. Defensins are cationic antimicrobialpeptides with a characteristic fold that is common to thetwo subfamilies, a- and b-defensins, despite a differencein the connectivity of their disulfide bonds [3]. Defen-sins are thought to exert broad microbial activity againstGram-positive and -negative bacteria, yeast, and someenveloped viruses [8]. Interestingly, the antimicrobialpeptide fails to display a cytolytic activity againsteukaryotic cells because of an electrostatic status of cellsurface [12]. In humans, a-defensins are expressed inneutrophils [5] and intestinal Paneth epithelial cells [10].In contrast, human b-defensins (hBD-1 to 4) are mostlyexpressed in various epithelial cells such as respiratoryand epidermal cells [21].

M. pneumoniae is a causative agent of acute respi-ratory illness, including tracheobronchitis and pneumo-nia [11], in humans. However, the mechanism by whichthe host protects against M. pneumoniae infection is notfully understood. Mycoplasmas have been shown to

cause a wide spectrum of direct or indirect effects onimmune cells [15]. Recently, macrophage-activatinglipopeptide-2, isolated from Mycoplasma fermentans,has been reported to activate macrophages by way ofToll-like receptor-2 (TLR2), resulting in production ofproinflammatory cytokines, including interleukin (IL)-1[14, 16]. However, the question of whether the antimi-crobial peptides are induced after M. pneumoniaeinfection remains to be elucidated.

Materials and Methods

Cell line. A human pulmonary squamous cell carcinoma, EBC-l cells(Cell Resource Center for Biomedical Research Institute ofDevelopment, Aging and Cancer, Tohoku University), wasmaintained as described previously [1].

Mycoplasma pneumoniae. M. pneumoniae FH and a M. pneumoniaeclinical isolate were cultured in pleuropneumonia-like organisms(PPLO) medium as described previously [1]. M. pneumoniae FH wascentrifuged for 30 minutes at 20,000 · g, and its pellet was heated at60�C for 30 minutes followed by lyophilization. The lyophilized M.pneumoniae was suspended at a concentration of 10 mg/mL (wt/vol) inphosphate-buffered saline and used for stimulation at a concentrationof 100 lg/mL.

Reagents. hBD-1, -2, and -3 were purchased from Peptide Institute(Osaka, Japan). Recombinant human IL-1b was obtained fromPeprotech EC (London, UK).Correspondence to: K. Kuwano; email: kuwano@med.kurume-u.ac.jp

CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 435–438DOI: 10.1007/s00284-005-0215-7 Current

MicrobiologyAn International Journal

ª Springer Science+Business Media, Inc. 2006

Antimicrobial assays. M. pneumoniae was diluted to 2 · 105/mL in10 mM sodium phosphate buffer (SPB) (pH 7.4) containing 0.03%Luria-Bertani broth. A 25-lL aliquot M. pneumoniae was incubatedwith 25 lL diluted peptide for 3 hours at 37�C as previously described[7]. The mixture of the M. pneumoniae and peptides was seriallydiluted 10-fold with SPB and plated on PPLO agar plates.Mycoplasmal colonies were enumerated the next day.

Reverse transcriptase-polymerase chain reaction analysis. EBC-1cells (2 · 106) were cultured in the presence of heat-inactivated M.pneumoniae and IL-1b at 50 ng/mL for 24 hours in 6-well plates. Thecells were collected and pelleted by centrifugation at 1,500 · g for 5minutes at 4�C. The pellets were used for RNA extraction. Total RNAwas extracted from the pellets by the method of Gough [6]. cDNAsynthesis and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed using an AMV Reverse Transcriptase XL andTaKaRa Ex Taq polymerase (Takara, Ohtsu, Japan) according to themanufacturer�s protocol. The primer sequences were as follows: hBD-1:5¢-GCTCAGCCTCCAAAGAAGCCA-3¢ and 5¢-CTTGCAGCACTTGGCCTTCCC-3¢; hBD-2: 5¢-CCAGCCATCAGCCATGAGGGT-3¢and 5¢-GGAGCCCTTTCTGAATCCGCA-3¢ [13]; hBD-3: 5¢-AGCCTAGCAGCTATGAGGATC-3¢ and 5¢-CTTCGGCAGCATTTTCGGCCA-3¢ [13]; b-actin: 5¢-AAGATGACCCAGATCATGTTTGAG-3¢and 5¢-AGGAGGAGCAATGATCTTGATCTT-3¢.

The PCR profile for hBD and b-actin included denaturation at94�C for 2 minutes followed by 35 cycles of denaturation at 94�C for30 seconds; annealing at 58�C (hBD-3 and b-actin), 59�C (hBD-1), or63�C (hBD-2) for 15 seconds; and extension at 72�C for 10 seconds.The PCR products were separated by electrophoresis on a 1.5% aga-rose gel and visualized by ethidium bromide staining.

Results and Discussion

Antimicrobial activity of hBD against M.pneumoniae. hBD-1, -2, and -3 were examined fortheir antimicrobial activity against M. pneumoniae. Asshown in Fig. 1, hBD-2 exerted antimicrobial activityagainst these strains in a dose-dependent manner at therange of 1 to 4 lM. At the concentration of 4 lM, thenumber of mycoplasmal colony was decreased by 100-to 1,000-fold compared with that of the control. Inaddition, hBD-3 showed a slightly higher microbialactivity than hBD-2 based on the efficient concentrationof peptides. In contrast, the antimicrobial activity ofhBD-1 was apparently lower than those of hBD-2 and -3as far as the concentration range was examined. Theresults show that hBD-2 and -3 possess antimicrobialactivity against M. pneumoniae.

The antimicrobial cationic peptides, including de-fensin, are known to show broad antimicrobial activity.To exert antimicrobial activity, the initial interactionbetween the positively charged amino acids, such asarginine and lysine, and the bacterial surface is of anelectrostatic nature caused by a multitude of negativelycharged groups on the surface of bacterial cells [17, 19].In contrast, the outer leaflet of the membranes ofeukaryotes is composed principally of lipids with no netcharge [12]. Therefore, eukaryote cells are lower in

susceptibility to antimicrobial cationic peptides thanprokaryote cells. To date, little is known about thesusceptibility of mycoplasmas to antimicrobial cationicpeptides. To our knowledge, this is the first reportshowing that mycoplasmas are susceptible to antimi-crobial peptides such as b-defensin. Intriguingly,mycoplasma membranes are composed of certain lipids,such as phosphatidylglycerol [2, 18], which are likely tocontain negatively charged moieties. Such moietieswould participate in the initial interaction betweenmycoplasma and the peptides.

Augmentation of hBD-2 mRNA in EBC-1 cells. Todetermine whether stimulation with M. pneumoniae andIL-1b induces the augmentation of hBD-1, -2, and -3mRNA expression, EBC-1 cells were stimulated withheat-inactivated M. pneumoniae and IL-1b. As shown inFig. 2, both M. pneumoniae and IL-1b augmented theexpression of hBD-2 mRNA several-fold. In addition,the level of hBD-1 mRNA expression was slightlyincreased after stimulation with M. pneumoniae and IL-1b, although the level was lower than that of hBD-2mRNA. Interestingly, the level of hBD-3 mRNAexpression was consistent even after the stimulation.The results show that both M. pneumoniae and IL-1bpossess the ability to augment the level of hBD-2 mRNAexpression in the EBC-1 cells.

When M. pneumoniae infects human lungs, par-ticularly tracheal epithelial cells, nonspecific defensemechanisms occur. The activation of macrophages byunknown components of mycoplasma causes produc-tion of proinflammatory cytokines, including IL-1 and

b

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Fig. 1. Antimicrobial activity of hBD. M. pneumoniae FH (a) and aM. pneumoniae clinical isolate (b) were incubated with diluted pep-tides for 3 hours at 37�C. The combinations between M. pneumoniaestrains and peptides were serially diluted 10-fold with SPB and platedon PPLO agar plates. Mycoplasmal colonies were enumerated thefollowing day. Solid triangle = hBD-1; solid square = hBD-2; solidcircle = hBD-3.

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TNF [1]. Subsequently, such inducible cytokines arethought to contribute in part to the pathogenesis ofM. pneumoniae infections [4, 9]. The proinflammatorycytokines are also known to be strong stimulators toactivate cells for gene expression. Recently, IL-1b wasreported to stimulate pulmonary cell line A549 toproduce hBD-2, indicating that the induced hBD-2might participate in the killing of invasive bacteria [22,23]. Similarly, heat-inactivated M. pneumoniae alsoupregulated the expression of hBD-2 mRNA (Fig. 2).Our preliminary data indicate that the component, de-rived from M. pneumoniae, responsible for inducinghBD-2 mRNA is likely to be characteristic of lipo-peptides (data not shown). The finding seems to upholdthe results of recent reports that a lipopeptide fromMycoplasmas, including M. pneumoniae, can activateNF-jB in human cells [16, 20].

In conclusion, human pulmonary squamous cell lineEBC-1, stimulated with M. pneumoniae and IL-1b, wasexamined for inducibility of hBD. HBD-2 was strikinglyupregulated by IL-1b as well as M. pneumoniae, butneither hBD-1 nor -3 was apparently upregulated. In anin vitro assay, hBD-2 was found to show highly anti-microbial activity against M. pneumoniae. Thus, ourresults suggest that inducible hBD-2 would play a role inthe protection of M. pneumoniae infection.

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aid for Scientific Re-search (C) from Japan Society for the Promotion of Science.

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β–actinhBD–1 hBD–2 hBD–3

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Fig. 2. Induction of hBD mRNA in EBC-1 cells. EBC-1 cells wereincubated with inactivated M. pneumoniae (a) and IL-1b at 50 ng/mL(b) for 24 h. Total RNA were isolated from the EBC-1 cells, and thenRT-PCR was performed as described in Materials and Methods.

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