Lipoic Acid Does Not Affect The Growth of Mycoplasma hominis Cells In Vitro

The species Mycoplasma hominis belongs to the genus Mycoplasma of the family Mycoplasmataceae. These bacteria are atypical bacteria because they do not have a cell wall, so they cannot be detected by the Gram-staining method, and their cells are uniquely small in size (Bébéar 2002; Krijnen et al. 2006). M. hominis is associated with various infections, mainly of the genitourinary system (such as cervicitis, pelvic inflammatory disease), and is responsible for infertility, obstetrical pathologies (premature delivery, premature rupture of membranes, chorioamnionitis), and neonatal infections (Koch et al. 1997; Waites et al. 2009; Bergin et al. 2017). Literature data indicate that M. hominis can cause also wound infections (Krijnen et al. 2006), meningitis (Zhou et al. 2016), post-operative infections (Whitson et al. 2014; Le Guern et al. 2015; Bergin et al. 2017; Qiu et al. 2017), and other disseminated infections in immunocompromised patients (Meyer et al. 1993; Miranda et al. 2005; Fernandez et al. 2017).

Lipoic acid (1,2-dithiolane-3-pentanoic acid; LA) and its reduced form, i.e., dihydrolipoic acid (DHLA; 6,8-dimercaptooctanoic) play a role as a cofactor in eukaryotes, most bacteria, and some archaea. LA exists mainly as lipoamide in the living cells, and it is attached with an amide linkage to the ε-amino group of a particular lysine residue on lipoyl carrier protein (LCP). As a cofactor of several enzymatic complexes, of which pyruvate dehydrogenase is best known, LA is an essential component of the energy metabolism of living organisms.

There are two mechanisms by which LCP becomes lipoylated in the living cells, namely, via de novo LA biosynthesis, which is an endogenous pathway, and via LA scavenging (or salvage) – an exogenous pathway. Eukaryotic organisms (including humans and other mammals) have essentially only one mechanism of this type, namely the de novo LA synthesis pathway (Spalding and Prigge 2010). So, the non-protein bound LA is gained by human and other animal’s cells from exogenous sources (drugs, dietary supplements, etc.). Due to its properties, LA is useful in treating numerous chronic and diet-related diseases (Salehi et al. 2019).

The situation is different in prokaryotic cells (but not all) where both of the mechanisms of LCP lipoylation mentioned above are present, that is de novo LA biosynthesis and LA scavenging (or salvage) – an exogenous pathway. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (the KEGG database is daily updated and freely available; http://www.genome.ad.jp/kegg/), it is known that, e.g., Escherichia coli, Bacillus subtilis, and Staphylococcus aureus have both pathways of acquiring LA: an endogenous pathway and LA salvage – an exogenous pathway. At this point, however, it should be noted that the results of some authors indicate that preparations containing LA also have antimicrobial activity (Zhao et al. 2018).

So the question arises whether LA is “a friend” to bacterial cells or their “enemy”? We know even less about the bacteria belonging to the family Mycoplasmataceae. The LA metabolic pathway in these cells is not well understood. It is unclear whether these cells have two pathways to metabolize LA or only one classical – de novo LA biosynthesis. There are no studies to solve this problem. The studies on the LA effect on bacteria multiplication and its bactericidal activity against these cells have not yet been published. The only paper that showed the antimicrobial activity of LA on bacterial cells belonging to the family Mycoplasmataceae (these were strains of Ureaplasma urealyticum and Ureaplasma parvum) was published by Biernat-Sudolska et al. (2020).

Considering the above, the aim of the present study was to determine for the first time whether LA influences the growth and multiplication of M. hominis strains. These studies are of a pilot nature.

The mean value of log CFU for control bacterial cultures was 6.90 (median 6.77, IQR (5.87; 7.74)), while the mean values of log CFU for bacteria cultured in the presence of LA at a concentration of 1,200 μg/ml, 120 μg/ml, and 12 μg/ml were 6.94 (6.87, (5.87; 7.78)), 6.86 (6.95, (5.81; 7.78)) and 7.17 (7.00, (6.38; 7.93)), respectively. These differences are not statistically significant (p = 0.807) (Fig. 1). Thus, the obtained results indicated that the presence of LA did not affect the growth of M. hominis.

Fig. 1.

Based on principal biochemical and microbiological knowledge, it should be hypothesized that LA should rather be “a friend” to bacterial cells than “an enemy” of them. It should be recalled that LA was isolated from bovine liver in 1950. The authors of this discovery – Reed and colleagues, describing their research, pointed out that the crystalline compound obtained turned out to be highly active against the growth of Streptococcus lactis, and also an important activator of the pyruvate dehydrogenase of Streptococcus faecalis. The researchers called the compound α-lipoic acid (Reed et al. 1951). The results of research published by Kafkewitz and colleagues in 1996 conducted on the selected strains of Pseudomonas were also interesting. The authors showed that the addition of a solution containing B vitamins (biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, niacin, pantothenic acid, and cyanocobalamin), p-aminobenzoic acid, and LA to the culture medium resulted in a 7–16% increase in the concentration of target compounds (2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl) degraded over the incubation period that was required for the concentration of the compound in the cultures to drop to approximately zero (Kafkewitz et al. 1996).

EA.hy 926 is a human cell line that exhibits highly differentiated functions characteristic of the human vascular endothelium. Inoculation of these cells with Rickettsia rickettsi resulted in a productive infection. Eremeeva and Silverman (1998) indicated that supplementing the culture medium with 100, 200, and 500 μM LA led to an increase in the viability of the infected EA.hy 926 cells after 96 hours to 45%, 51%, and 70%, respectively, compared with 26% for the samples untreated with LA.

Zorzoli et al. (2016) also obtained exciting results in the murine sepsis model, who in the murine sepsis model due to S. aureus infection found that de novo biosynthesis or salvage of LA promoted S. aureus survival. When both LA biosynthesis and salvage pathways were blocked, then S. aureus was rendered avirulent.

Several authors showed the second bacterial-unfriendly “face” of LA. The research of Jariwalla et al. (2008) indicated that supplementation with LA acid might positively impact patients with HIV and acquired immune deficiency syndrome. In addition, it is worth mentioning the preprint by Zhong et al. (2019) on the clinical efficacy and safety of LA for critically ill patients with coronavirus disease 2019 (COVID-19).

Nevertheless, the antiviral activity of LA seems less surprising than its antibacterial activity because LA is an integral part of the energy metabolism of living organisms, including bacteria, as mentioned before. Noteworthy, there are over a dozen studies whose results have revealed the antibacterial properties of LA. We present a few of them here. Among the new methods of antimicrobial therapy, particular consideration should be given to antimicrobial peptides (AMP), including the Bac8c (RIWVIWRR-NH2), the natural AMP exhibiting a high antibacterial activity against Gram-negative and Gram-positive bacteria. Zhou et al. (2020) used LA as a fatty acid hydrophobic ligand to modify Bac8c (LA-Bac8c). The authors reported that minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays showed that LA-Bac8c exhibited lower MIC and lowered MBC values against S. aureus and methicillin-resistant S. aureus (MRSA) than Bac8c. The authors also demonstrated that LA-Bac8c showed better activity against S. aureus and MRSA biofilms, which have been formed or are being formed, than Bac8c (Zhou et al. 2020). It was also shown that LA exerted moderate inhibitory effects against Cronobacter sakazakii strains (Shi et al. 2016).

In our previous research on Ureaplasma urealyticum and Ureaplasma parvum, we observed that LA only at a concentration of 1,200 μg/ml had a statistically significant (p < 0.001) inhibitory effect on cell division of Ureaplasma strains compared to the control (Biernat-Sudolska et al. 2020).

So, what is the mechanism by which LA displays its “Janus face” in microorganisms? Our hypothesis assumes involvement of sulfane sulfur. Sulfane sulfur is a divalent, reactive, and labile sulfur atom covalently bonded to another sulfur. This form of sulfur, with six valence electrons, occurs in the oxidation state of 0 or –1, and quickly leaves the compound being transferred to various acceptors, including the thiol groups (-SH) of the cysteine residues of proteins. This is another method of covalent modification of proteins, changing their biological activity (Iciek et al. 2018). It is known that in bacteria, the concentration of sulfane sulfur can be higher than 100 μM (Ran et al. 2019). It has recently been demonstrated that sulfane sulfur increases the virulence in Pseudomonas aeruginosa PAO1 by targeting its quorum sensing (QS) systems (Xuan et al. 2021). However, we recall the long known antimicrobial activity of garlic, for which, as we know today, diallyl sulfides are responsible, which are the source of sulfane sulfur. For example, it is known that diallyl disulfide (DADS) from garlic reduces the pathogenicity and biofilm development of P. aeruginosa PAO1 by targeting its QS systems (Li et al. 2018) “Cognitive dissonance”? Not necessarily. The concentration of DADS used by the researchers cited above was 1.28 mg/ml (about 8.8 mM). Hou et al. (2019), in studies on E. coli cells, showed that at high concentrations, sulfane sulfur was toxic to bacteria. Why did we devote so much attention to the problem of sulfane sulfur? Since, in our opinion, the biological activity of LA is also associated with the generation of sulfane sulfur compounds (Bilska et al. 2008; Bilska-Wilkosz et al. 2017).

The second problem that arises when studying the antibacterial properties of LA is its metabolism in these cells, which was mentioned in the Introduction. Unfortunately, the LA metabolic pathway in Mycoplasma species cells is not well understood. It is unclear whether these cells have two pathways to metabolize LA. The available data suggest that, like eukaryotic organisms, Mycoplasma cells do not have the LA salvage pathway. It would mean that the presence of LA in the medium should not affect the growth of these bacterial cells. The first research on this problem dates back to the 1960s. The results obtained at that time showed that although the presence of LA in the medium stimulated the growth of Mycoplasma strain Y in primary culture, on the other hand, LA had little or no effect on its growth in subculture in the same medium (Rodwell 1969).

Based on our results, none of the above hypotheses can be reliably confirmed or rejected because our data did not reach statistical significance. We did not obtain statistical significance because for 52.5% of M. hominis strains, the CFU increased for all tested concentrations of LA. It could indicate that M. hominis cells do not have the LA salvage pathway. In opposite, at concentrations of 1,200 μg/ml and 120 μg/ml, a decrease of the CFU was observed for 45% of the tested strains (Fig. 2, Table I). After averaging these values and subjecting the results to statistical analysis, it turned out that LA does not affect the growth of M. hominis cells in in vitro studies.

Fig. 2.

The problem is interesting but requires further research. Perhaps this research should be repeated on the selected strains of M. hominis. It seems, however, that the research using the molecular system able to knock out a gene and/or genes relevant to de novo LA biosynthesis pathway is needed for a definite answer to this problem. Further studies would also need to show that LA generates sulfane sulfur in M. hominis cells and affects the mechanisms and/or structures necessary for cell survival and/or growth. Thus, the study presented here is a modest contribution and an invitation to research on the influence of LA on bacteria from the Mycoplasmataceae family. This type of research can provide several answers to questions important for a biochemist, microbiologist, and physician. Taken together, this research is an introduction to further investigations aimed at a better understanding of LA metabolism in the microbial cells, which is also essential for the search for new antimicrobial drugs.