Staphylococcal secretory inhibitor of platelet microbicidal protein is associated with prostatitis source

A number of micro-organisms are able to infect the reproductive tract tissues in humans with serious consequences for reproductive function (Domingue & Hellstrom, 1998; Everaert et al., 2003). A common result of microbial infection of the reproductive tract is prostatitis, a condition that may lead to transient or permanent infertility (Schoor, 2002; Everaert et al., 2003). Bacterial infection of the prostate may occur as a result of ascending urethral infection or by reflux of infected urine into prostatic ducts emptying into the posterior urethra (Terai et al., 2000). Other possible routes of prostatitis include invasion of bacteria by lymphogenous or haematogenous spread (Domingue & Hellstrom, 1998). Chronic bacterial prostatitis (CBP) is a subtle illness, characterized by the persistence of bacteria in the prostatic secretory system (Schoor, 2002; Everaert et al., 2003). The most common causative agents of CBP are coagulase-negative staphylococci (CNS) and Staphylococcus aureus (Lowentritt et al., 1995; Domingue & Hellstrom, 1998; Ivanov, 2005). As it is difficult to establish precisely the significance of various micro-organisms in the pathogenesis of CBP, it is imperative to delineate both microbial and host factors that contribute to its development (Domingue & Hellstrom, 1998; Schoor, 2002; Hua et al., 2005).

The major role of endogenous cationic antimicrobial peptides in preventing the onset of infection has been emphasized recently (Hancock & Chapple, 1999; Dürr & Peschel, 2002). Such peptides have also been found by several authors in human platelets and are designated ß-lysins (Donaldson & Tew, 1977), thrombocidins (Krijgsveld et al., 2000), thrombodefensins (Dankert, 1988) and platelet microbicidal protein (PMP) (Yeaman, 1997; Ivanov, 2005). These peptides are secreted at sites of infection and exert microbicidal activity against many pathogens, including Gram-positive cocci. Dankert et al. (1995) demonstrated that thrombodefensins are an important defence factor against viridans streptococcal endocarditis. Wu et al. (1994) showed that resistance in vitro of clinical staphylococcal and viridans group streptococcal strains to rabbit PMP correlated with the endovascular infectious source. The results of several studies (Dhawan et al., 1997; Kupferwasser et al., 2002) have shown that susceptibility to PMP is associated with staphylococcal endocarditis, and Kupferwasser et al. (1999) demonstrated that the staphylococcal multidrug-resistance gene qacA also mediates resistance to PMP in vitro.

In a recent publication, we showed that resistance of urethral staphylococcal and enterococcal strains to human PMP in vitro correlated with the diagnosis of CBP (Ivanov, 2005). However, it is surprising that an extracellular bacterial product demonstrating this remarkable anti-PMP potential has not been described. The purpose of this study was to define the existence of an extracellular substance, designated secretory inhibitor of PMP (SIPMP), detectable in cell-free staphylococcal culture supernatants, that displays hitherto unrecognized PMP-consuming properties. In this paper, we also report the detection in vitro of SIPMP phenotypes of urethral staphylococcal isolates, along with a comparison with isolates from patients with or without CBP.

Preparation and standardization of PMP. PMP was prepared as described previously (Ivanov, 2005). Briefly, healthy human platelet-rich plasma was obtained by low-speed centrifugation of 1 day-outdated platelets from the Blood Donor Department of the Orenburg Regional Medical Center. The platelet-rich plasma was then dispensed into polypropylene tubes and centrifuged again at 250 g for 30 min at 25 °C. The supernatants were removed and centrifuged at 1000 g for 30 min at 25 °C and the sedimented platelets were washed three times with Tyrode's buffer (138 mM NaCl, 3.6 mM KCl, 10 mM NaHCO₃, 0.4 mM NaH₂PO₄, 10 mM MgCl₂, 6 mM glucose, adjusted to pH 7.3 with phosphoric acid). The washed platelets were suspended in 5 vols ice-cold 30 % acetic acid and stirred in melting ice for 24 h. The resulting extract was centrifuged at 10 000 g for 15 min and the PMP-rich supernatant was recovered. PMP preparations were stored at 20 °C and used for subsequent studies within 30 days. PMP bioactivity assays were performed with Bacillus subtilis ATCC 6633, an indicator organism highly sensitive to the bactericidal action of PMP (Donaldson & Tew, 1977). Exposure of 10⁸ washed human platelets ml¹ to 5 ml ice-cold 30 % acetic acid resulted in mean supernatant protein concentrations of ∼70 mg ml¹. The PMP susceptibility of bacterial strains was determined by exposing bacterial cells to serial dilutions of PMP. B. subtilis was grown in tryptic soy broth (TSB) at 37 °C for 18 h; organisms were harvested by centrifugation, washed twice in PBS (pH 7.2) and resuspended in PBS prior to use. Bacteria were diluted to 10⁴ c.f.u. ml¹ in PBS. Serial dilutions of PMP were prepared in PBS and 900 µl PMP aliquots were transferred to low-protein-binding protein microtitre tubes (Costar Glass Works). To each of the tubes, 100 µl bacterial suspension was added. The tubes were incubated on a rotary shaker (300 r.p.m.) at 37 °C. After 1 h, aliquots of 200 µl were plated on blood agar plates. The microbicidal activity of PMP was assessed the next day after counting colonies on the agar plates and defined as the concentration of protein that retained 50 % lethality for B. subtilis (Hirsch, 1960). The bioactivity of such PMP preparations against B. subtilis ranged from 0.5 to 1.0 µg ml¹. Control samples were found to possess no anti-B. subtilis bioactivity.

Detection of PMP resistance and SIPMP production. Well-characterized urethral isolates of S. aureus (n=24) and CNS (Staphylococcus epidermidis, n=27; Staphylococcus hominis, n=20) from patients with or without CBP were kindly provided by Serge Cherkasov (Orenburg State Medical Academy, Orenburg, Russia). The determination of patients and cases from which the isolates were initially obtained as CBP and non-CBP was made by the contributing investigator, using standard clinical parameters, prior to knowledge of an isolates SIPMP production and PMP susceptibility. The bacteria were cultured overnight in TSB and cell-free supernatants were obtained by centrifugation. Staphylococcal supernatants were sterilized by filtration through 0.45 µm pore size membranes (Millipore). Each culture supernatant (0.6 ml) (an equal volume of TSB was loaded in the control tubes) was combined with 0.3 ml PMP at 3.0 µg ml¹ and incubated at 37 °C. After 1 h, 100 µl B. subtilis suspension at 10⁴ c.f.u. ml¹ was added to each of the tubes. The tubes were incubated on a rotary shaker (300 r.p.m.) at 37 °C. After 1 h, aliquots of 200 µl were plated on blood agar plates. Colonies were counted after overnight incubation at 37 °C and the numbers of surviving micro-organisms were calculated. SIPMP production was expressed as the percentage inhibition of PMP bactericidal activity and calculated using the formula: percentage inhibition=(NoNk1)/(Nk2Nk1)x100, where No was the number of surviving B. subtilis cells in the presence of staphylococcal supernatant and PMP, Nk1 was the number of surviving B. subtilis cells in the presence of PMP alone, and Nk2 was the number of surviving B. subtilis cells in TSB.

The PMP susceptibility of bacterial strains was determined by exposing 2x10³ bacterial cells to serial dilutions of PMP from 5 µg ml¹ to 15 µg ml¹, as described by Yeaman et al. (1992). To define the proportion of strains considered PMP-susceptible and PMP-resistant, a survival of 50 % of the initial inoculum at a PMP concentration of 5 µg ml¹ was considered to be a relative PMP-susceptibility breakpoint, as in the data of Wu et al. (1994).

Statistical analyses were performed using Student's t-test. Correlation between SIPMP production and PMP resistance was calculated as r² values by the Pearson correlation calculation using Microsoft Excel. Values of P0.05 and r²0.5 were considered to be significant.

Among the 24 S. aureus isolates studied, 13 urethral isolates were from patients with CBP, whilst 11 isolates were from patients without CBP (Table 1). Of the CBP strains tested, 10/13 (76.9 %) were considered to be resistant to the bactericidal action of PMP compared with only 2/11 of the non-CBP isolates (18.2 %, P<0.001). Furthermore, 3/13 (23.1 %) of CBP strains were resistant to 15 µg PMP ml¹ (Table 2).

Table 1. PMP susceptibility and SIPMP production of urethral staphylococcus isolates

Table 2. PMP susceptibility of non-CBP and CBP isolates

Of the 47 urethral CNS isolates tested, 29 and 18 were from CBP and non-CBP cases, respectively. A significantly higher proportion of CBP strains of CNS (93.1 vs 5.6 %) was resistant to PMP compared with non-CBP strains (Table 1; P<0.001). Moreover, all non-CBP strains exhibited either low-level resistance or susceptibility to 10 µg PMP ml¹. However, 16/29 CBP strains (55.2 %) were resistant to higher bactericidal concentrations of PMP (15 µg ml¹).

Among the SIPMP-producing strains, organisms isolated from patients without CBP had mean SIPMP production levels of 10.3±1.2 and 13.25±1.72 % for S. aureus and CNS, respectively (Table 3). In contrast to bacteria isolated from the control group, strains isolated from men with CBP demonstrated more intensive inhibition of the bactericidal activity of PMP. The culture supernatants of S. aureus from the CBP group were significantly more active in decreasing levels of PMP-induced killing of B. subtilis (23.38±4.2 %, P<0.05). CBP strains of CNS demonstrated SIPMP production at a mean level of 23.69±1.87 % (P<0.01). SIPMP production correlated with staphylococcal resistance to PMP (r²=0.6082 and 0.7264 for S. aureus and CNS, respectively).

Table 3. SIPMP production of urethral staphylococcus isolates

At local sites of microbial infection, platelets, neutrophils and macrophages release large amounts of different bactericidal peptides (Dürr & Peschel, 2002). CBP is characterized by recurrent urinary tract infections and persistence of bacteria in the prostatic secretory system, despite the presence of multiple antibacterial peptides in prostatic fluid (Fair & Parrish, 1981; Com et al., 2003). There is an urgent need to understand the virulence properties of bacteria that are associated with chronic infection of the prostate. Identifying such factor(s) would be helpful in devising effective treatment strategies.

In the present work, we detected an extracellular staphylococcal product with remarkable anti-PMP potential, which, to our knowledge, has not been described before. We anticipate that this substance represents a novel determinant of staphylococcal pathogenicity, as it probably causes local inhibition of the bactericidal action of PMP. As PMP may play an important role in the killing of staphylococci and preventing the onset of bacterial infection of the prostate, we speculated that SIPMP might enhance the infectivity of these bacteria through the abortive consumption of PMP in the fluid phase. The strategy underlying this process would be straightforward and effective. We believe that SIPMP represents a widespread and hitherto unrecognized determinant of bacterial pathogenicity. Similarly, in a study of the distribution of streptococcal inhibitor of complement variants in pharyngitis and invasive isolates by Hoe et al. (2001), 62 % of group A streptococci from patients with pharyngitis produced this extracellular protein. In addition, a recent study by Fernie-King et al. (2002) showed that purified secretory inhibitor of complement could block two additional components of the immune system: lysozyme and secretory leukocyte proteinase inhibitor. Collectively, our study and the results of several other studies (Bukharin et al., 1996; Fernie-King et al., 2002; Deckers et al., 2004; Langley et al., 2005) suggest that inactivation of components of innate immunity may be important for bacterial pathogens to induce and perpetuate chronic infections of different localization by surviving or avoiding microbicidal protein-mediated clearance.

Data from the present study may have significant implications for the understanding of the pathogenesis of CBP, as well as for future improvements in the prevention and therapy of CBP. Whether or not SIPMP is truly important in pathogenicity may become evident in the future from in vivo studies with selected SIPMP-positive and SIPMP-negative strains.

This study was supported by grant 04-04-97508 from the Russian Fund of Basic Research.