Oxyrase TM for Optimal Growth of Trichomonas vaginalis Isolates

The inability of some fresh clinical isolates of Trichomonas vaginalis to grow and multiply when inoculated into the In-Pouch TM trichomonas growth medium followed by death upon inoculation into the trypticase-yeast extract-maltose (TYM)-horse serum complex medium was investigated. Of fifty-one fresh clinical isolates derived from patients with trichomonosis, 11 (~20%) failed to grow to numbers after passage into the complex medium sufficient for cryopreservation (labeled Group 1). Isolates labeled Group 2 (~80%) thrived in both the In-Pouch TM and complex media. Experiments using the Oxyrase TM scavenging system to remove oxygen from the medium revealed that Group 1 isolates grew to numbers equal to Group 2 isolate trichomonads. Group 1 isolates consisted of both dsRNA virus-negative and positive isolates, showing that absence or presence of virus was not responsible for the growth characteristics of the Group 1 isolate organisms. This further indicated that the Group 1 isolate parasites are unable to survive in a microaerophilic environment compared to Group 2 isolate trichomonads, suggesting that the patient vaginal environment was anaerobic. Analysis by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total proteins of to compare Group 1 and Group 2 organisms showed no differences in protein patterns. Likewise, both Group 1 and Group 2 isolate parasites had similar total proteinase patterns by substrate gel analysis after electrophoresis. Comparative studies of the property of cytoadherence and amounts of adhesins also showed identical results for Group 1 organisms grown in Oxyrase TM and Group 2 parasites in the presence or absence of Oxyrase TM . These results are discussed in relation to the patient’s vaginal anaerobic environment as critical for establishment of infection by Group 1 isolate parasites. This work now allows for investigators to perform studies of T. vaginalis isolates heretofore unable to be cultured and cryopreserved.


Introduction
Trichomonas vaginalis causes trichomonosis, the number one, non-viral and curable sexually transmitted infection (STI) worldwide [1]. This STI is responsible for numerous adverse health outcomes for women [2]. Numerous reports have established differences in virulence properties among trichomonads cultured in vitro over extended periods versus fresh clinical isolate organisms.
For example, freshly-derived clinical isolate organisms grown in batch culture for three weeks lose the iron-upregulated synthesis of adhesins that mediate cytoadherence, a property preparatory for infection [4][5][6]. Likewise, the dsRNA viruses that infect some T. vaginalis isolates are lost after several weeks after batch cultivation in complex medium [6,7]. These and other observations make evident the need to be able to collect and preserve fresh clinical isolates to analyze and understand the properties important to the hostparasite interrelationship and to be able to identify trichomonad virulence factors. Such a knowledge base is prerequisite to any future interference strategies for this significant STI.
In this study the hypothesis that some T. vaginalis isolates that are difficult to grow to optimal density levels for cryopreservation was the result of the level of oxygen in the growth medium. The results from growth experiments showed that eleven of fifty-one (~20%) trichomonal isolates (labeled Group 1) freshly-derived from patients were lost after inoculation of the In-Pouch TM trichomonal medium and the TYM-serum complex media [8]. This finding is in contrast with the forty (~80%) isolates called Group 2 that grew in both the In-Pouch TM and the complex media. There are numerous reports of microorganisms requiring removal of oxygen from the growth medium by Oxyrase TM for optimal growth and multiplication and for performing experiments [9][10][11]. Therefore, this study on the Group 1 isolate trichomonads show that addition of Oxyrase TM to the In-Pouch TM and the complex media [8] permitted achieving optimal cell densities equal to the Group 2 isolates, and these isolate organisms were able to be cryopreserved. The significance of these findings regarding the ability to cryopreserve in vitro growth-resistant fresh clinical isolates and the patient vaginal environment that is permissive for growth in vivo of these parasites is discussed. supplemented with 10% heat-inactivated horse serum (complex medium) [8,12]. A duplicate T-25 flask with complex medium was supplemented with 0.6ml Oxyrase TM (Oxyrase, Inc., Mansfield OH, USA) (0.1ml per 5ml of medium) to remove oxygen as per the manufacturer's instructions. Numbers of organisms were obtained daily for up to 7days using a hemacytometer. Isolates are referred to as Type I and Type II indicating the absence and presence of dsRNA virus, respectively, that infect some fresh clinical isolates and the long term grown NYH 286 isolate trichomonads [6,7].

Total trichomonad proteins and protease analyses and levels of cytoadherence and amounts of adhesins
Total protein preparations of T. vaginalis proteins for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained by trichloroacetic acid precipitation as described before [12]. Conditions for one dimensional SDS-PAGE using 7.5% acrylamide gels and Coomassie Brilliant blue-staining of gels for comparing total proteins of trichomonads grown in the absence and presence of Oxyrase TM was performed using established procedures [12]. The proteases of T. vaginalis organisms were analyzed by SDSPAGE that was performed with acrylamide copolymerized with gelatin as substrate as described previously [13].
The levels of cytoadherence to vaginal epithelial cells and the amounts of the specific adhesin proteins that mediate this property of attachment to host cells were also examined for Group 1 parasites grown in OxyraseTM medium and compared with the Group 2 organisms, as before [3][4][5]14]. Table 1 presents the summary of the freshly-derived isolate trichomonads assigned to Group 1 and Group 2 based on the requirement of Oxyrase TM to achieve optimal cell densities. A total of fifty-one isolates were examined, and eleven isolates (~20%) were found to require Oxyrase TM for growth (Group 1) compared to the forty isolates (~80%) without an Oxyrase TM requirement (Group 2). Further, as shown in Table 1, 50% of isolates were either positive or negative for the presence of the dsRNA virus as reported before [6,7], and the data show that the dsRNA virus had no effect on the growth kinetics of the organisms (Table 1).  [6,7]. These data show the requirement of removing oxygen from the growth medium for optimal growth and multiplication of Group 1 isolate organisms.   absence (solid circles) of Oxyrase TM in the growth medium. As can be seen in Figure 3, the batch culture in the complex medium under both conditions were nearly identical. It is noteworthy that for long term-grown isolate organisms, the high generation times for optimal densities were achieved by day two compared with the fresh clinical isolates of both Group 1 and 2 isolate trichomonads ( Figure 3).  All isolates are freshly-derived clinical isolates except for isolate NYH 286, which is a laboratory-adapted isolate that has been passaged over many years in batch culture. The preparation of total trichloroacetic acid-precipitated proteins of T. vaginalis is as previously published [12].

Examination of total proteins and proteinases of Group 1 and Group 2 parasites
The Coomassie Brilliant, blue-stained gel of total proteins of two Group 1 and four Group 2 isolate trichomonads after SDS-PAGE is presented in Figure 4. To show that the dsRNA virus did not affect the protein patterns for both Groups 1 and 2, four isolates infected with the dsRNA virus and two isolates without the virus were analyzed. Each isolate was grown in the absence (lanes labeled C) and presence (lanes labeled Oxy) of Oxyrase TM . For the Group 1 isolates without Oxyrase TM with low numbers of organisms as shown in Figure 1, inoculation of multiple T-25 flasks was required to obtain the numbers necessary for total protein preparations as detailed in an earlier report [12]. As can be seen, except for the intensity of staining for some protein bands, the one-dimensional total protein patterns were nearly identical. These findings show that both the growth in Oxyrase TM and the presence or absence of the dsRNA virus did not affect the protein composition of these Group 1 and 2 isolate parasites ( Figure 4).
We then analyzed by substrate gel electrophoresis the total proteinases of the Oxyrase TM -grown Group 1 and Group 2 isolate parasites, as before [13]. As with the total protein patterns shown above, there were no qualitative and quantitative differences in proteinases for organisms of either Group (data not shown).
Finally, we also performed standard adherence assays of the Group 1 and Group 2 isolate trichomonads grown in the presence of Oxyrase TM using vaginal epithelial cells, as before [3][4][5]14]. Not unexpectedly for fresh isolate organisms both Group 1 and Group 2 organisms had similarly high levels of adherence and amounts of the adhesin proteins [3][4][5]14]. Also, as expected for fresh clinical isolates [14,15], the organisms for both Group 1 and Group 2 had up-regulated expression of the adhesin proteins when compared to the long term-grown NYH 286 isolate [3,4]. Collectively, these data indicate that, after correcting for the ability to grow in the TYMserum complex medium via addition of Oxyrase TM , all T. vaginalis trichomonads of both Group 1 and Group 2 were identical for the properties examined.

Discussion
The discovery of the Oxyrase TM enzyme system derived from the E. coli cytoplasmic membrane that is efficient for scavenging oxygen in broth medium has been shown to enhance the growth of microorganisms [8][9][10]. Therefore, this study tested the hypothesis that oxygen in the growth medium for T. vaginalis restricted growth and multiplication of trichomonads of some fresh clinical isolates.
These isolates (labeled Group 1) were unable to thrive in vitro and were subsequently lost when compared to Group 2 isolates with excellent growth kinetics that permitted cryopreservation. The data presented in Figure 1 indeed show that removal of oxygen from the growth medium by Oxyrase TM permitted levels of growth and multiplication of Group 1 organisms equivalent to those observed for Group 2 parasites ( Figure 2). were analyzed for total protein profiles ( Figure 4) [12], proteinases [13] and levels of cytoadherence and amounts of adhesin proteins

Conclusion
This report shows for the first time that some fresh clinical T.
vaginalis isolates require the removal of oxygen from the growth medium for optimal growth necessary for cryopreservation. This is important to be able to delineate and understand the biological properties, such as expression of proteins [12] and cytoadherence to vaginal epithelial cells [5], and virulence factors, such as adhesins [3,4] and proteinases [13], preparatory to infection of humans.