Inflammation-inducing Factors of Mycoplasma pneumoniae

Mycoplasma pneumoniae, which causes mycoplasmal pneumonia in human, mainly causes pneumonia in children, although it occasionally causes disease in infants and geriatrics. Some pathogenic factors produced by M. pneumoniae, such as hydrogen peroxide and Community-Acquired Respiratory Distress Syndrome (CARDS) toxin have been well studied. However, these factors alone cannot explain this predilection. The low incidence rate of mycoplasmal pneumonia in infants and geriatrics implies that the strong inflammatory responses induced by M. pneumoniae coordinate with the pathogenic factors to induce pneumonia. However, M. pneumoniae lacks a cell wall and does not possess an inflammation-inducing endotoxin, such as lipopolysaccharide (LPS). In M. pneumoniae, lipoproteins were identified as an inflammation-inducing factor. Lipoproteins induce inflammatory responses through Toll-like receptors (TLR) 2. Because Mycoplasma species lack a cell wall and lipoproteins anchored in the membrane are exposed, lipoproteins and TLR2 have been thought to be important for the pathogenesis of M. pneumoniae. However, recent reports suggest that M. pneumoniae also induces inflammatory responses also in a TLR2-independent manner. TLR4 and autophagy are involved in this TLR2-independent inflammation. In addition, the CARDS toxin or M. pneumoniae cytadherence induces inflammatory responses through an intracellular receptor protein complex called the inflammasome. In this review, the inflammation-inducing factors of M. pneumoniae are summarized.


INTRODUCTION
Mycoplasma pneumoniae causes primary atypical pneumonia, tracheobronchitis, pharyngitis, and asthma in humans (Gil et al., 1993;Kraft et al., 1998;Waites and Talkington, 2004). The age distribution of patients with pneumonia caused by M. pneumoniae is characteristic. The incidence is highest among school-aged children and young adults and lower in infants and geriatrics (Denny et al., 1971;Foy et al., 1979). Some pathogenic factors of M. pneumoniae, such as hydrogen peroxide, Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, and nuclease, have been reported to be associated with the development of pneumonia (Somerson et al., 1965;Cohen and Somerson, 1967;Kannan and Baseman, 2006;Hames et al., 2009;Somarajan et al., 2010). However, these pathogenic factors are insufficient to explain the age distribution of patients with pneumonia caused by M. pneumoniae. Generally, the immune system in infants and geriatrics is immature compared with that in young adults. The symptoms of pneumonia caused by M. pneumoniae are correlated with the induction of pro-inflammatory cytokines (Tryon and Baseman, 1992;Salvatore et al., 2007). These findings suggest that the excessive immune responses induced by M. pneumoniae play an important role in the development of pneumonia. In this review, the molecular mechanisms of inflammation induced by M. pneumoniae are summarized (Table 1).

Lipoproteins of Mycoplasma Species
It has been reported that some Mycoplasma species induce pro-inflammatory cytokines and stimulate various immune cells (Atkin et al., 1986;Kirchner et al., 1986;Teh et al., 1988). Because Mycoplasma species are devoid of a cell wall and lack immune cell stimulator such as lipopolysaccharide (LPS) or peptidoglycan (Mizel et al., 1978;Staber et al., 1978), the factors responsible for the induction of inflammatory responses have been unclear for a long time. The first report on the inflammation-inducing factor of Mycoplasma species was published by Quentmeier et al. (1990). They reported that a high-molecular-weight (HMW) protein of M. fermentans known as MDHM possesses interleukin (IL)-6-inducing activity in macrophages. Because the activity of MDHM was resistant to proteinase K, the active component of MDHM was thought to be a low-molecular-weight compound. In 1996, Muhlradt et al. identified the active component of MDHM as S-(2,3-dihydroxypropyl) cysteine (Muhlradt et al., 1996). This component was similar to the N-terminal structure of an Escherichia coli-derived lipoprotein identified in 1969 by Braun et al. Braun (1975). Muhlradt et al. (1997) also purified the inflammation-inducing factor from M. fermentans culture and demonstrated that the active component is the diacylated lipopeptide, S-(2,3-bisacyloxypropyl)-CGNNDESNISFKEK. They named it macrophage-activating lipopeptide-2 (MALP-2). After these reports, inflammationinducing lipoproteins were purified and identified in various Mycoplasma species (Jan et al., 1996a;Muhlradt et al., 1997Muhlradt et al., , 1998Shibata et al., 2000), including M. pneumoniae (Shimizu et al., 2005).

Structure of Lipoprotein and TLR
Lipoproteins were discovered in 1969 by Braun et al. (Braun, 1975). Lipoproteins are hydrophilic membrane proteins characterized by a conserved N-terminal lipid-modified cysteine residue. Lipoproteins contain S-glyceryl cysteine modified with three fatty acids (N-acyl-S-diacylglyceryl cysteine) at their N-terminal. This triacylated structure is also called Braun's lipoprotein. Braun's Lipoproteins are synthesized by the following three steps (  (3) Linkage of an acyl chain to the amino group of the amino-terminal cysteine (N-acylation) by prolipoprotein N-acyl-transferase (Lnt). Because genes orthologous to Lnt gene are not found in some bacterial species (Firmicutes and and Tenericutes), including Mycoplasma species, lipoproteins from these bacterial species have been assumed to be of the diacylated form (Nakayama et al., 2012). Toll-like receptors are a type of pattern-recognition receptors that play critical roles in early innate recognition and host inflammatory responses against invading microbes (Kopp and Medzhitov, 1999;Akira and Takeda, 2004). Among the 11 reported TLR family members, TLR2 plays a central role in the recognition of lipoproteins. TLR2 recognizes the S-diacylglyceryl cysteine portions of both diacylated and triacylated lipoproteins (Botos et al., 2011). TLR1 coordinate with TLR2 to recognize triacylated lipoproteins through the recognition of the third acyl chain amide-linked to the cysteine using a hydrophobic pocket within TLR1 (Jin et al., 2007). However, TLR2 alone is not able to recognize diacylated lipoproteins. TLR6 assists in the recognition of diacylated lipoproteins by associating with the amino acid portion of diacylated lipoproteins (Kang et al., 2009). Although there are some exceptions (Buwitt-Beckmann et al., 2005;Kurokawa et al., 2009), diacylated and triacylated lipoproteins are generally recognaized by TLR2/6 and TLR2/1 heterodimers, respectively.
The presence of triacylated lipoproteins in Mycoplasma species has been controversial. The lipoproteins from M. fermentans, M. hyorhinis, M. salivarium, and M. gallisepticum have been shown to be diacylated lipoproteins and not N-acylated (Jan et al., 1996a;Muhlradt et al., 1997Muhlradt et al., , 1998Shibata et al., 2000). The Lnt gene, which encodes the enzyme responsible for N-acylation has not been found in any mycoplasma genomes (Fraser et al., 1995;Himmelreich et al., 1996;Sasaki et al., 2002). However, a study on the ratio of N-amide and O-ester bonds in M. gallisepticum and M. mycoides suggested the presence of triacylated lipoproteins (Jan et al., 1996b). Furthermore, the resistance to Edoman degradation of proteins from M. mycoides also indicated the presence of N-acylation (Chambaud et al., 1999). These data imply the presence of triacylated lipoproteins in Mycoplasma species.

Lipoproteins of M. pneumoniae
Lipoproteins of M. pneumonie are summarized in Table 2. In M. pneumoniae, Shimizu et al. (2005) reported that the subunit b of the F 0 F 1 ATP synthase (MPN602) is a diacylated lipoprotein that induces inflammatory responses through TLR2. Into et al. (2007) also reported that M. pneumoniae has 48 lipoproteins and that the common N-terminal structure of these lipoproteins induces inflammatory responses. Interestingly, Some of these lipoproteins (MPN162, MPN611) were recognized by TLR1 and TLR2, suggesting that M. pneumoniae contains triacylted lipoproteins (Shimizu et al., 2007). Kurokawa et al.

M. pneumoniae and Autophagy
Because Mycoplasma species lack cell walls, they do not contain immunostimulants such as LPS, peptidoglycan, or lipoteichoic acid. Therefore, lipoproteins seem to be key factors in M. pneumoniae-induced inflammatory responses and to facilitate the development of pneumonia in humans. However, the existence of lipoproteins in non-pathogenic Mycoplasma species suggests the presence of an alternative mechanism by which M. pneumoniae induce inflammatory responses.
Autophagy is a cellular response that involves the sequestration of regions within the cytosol with double membrane compartments. Autophagy has been shown to play important roles in the cellular response to starvation, cell death, removal of damaged organelles, and neurodegenerative diseases (Levine, 2005). It has recently been recognized that autophagy is involved in both innate and adaptive immunity against various microorganisms (Schmid and Munz, 2007;Deretic et al., 2013;Ma et al., 2013).
Recently, Shimizu et al. demonstrated that M. pneumoniae induces strong inflammatory responses, even in macrophages derived from TLR2 knockout (KO) mice (Shimizu et al., 2014). M. pneumoniae internalized into macrophages through phagocytosis were co-localized with the autophagosome, and autophagy inhibitors decreased the induction of proinflammatory cytokines, suggesting the autophagy-mediated induction of inflammatory responses. Because this TLR2independent induction was inhibited in macrophages derived from TLR2/4 double KO mouse, TLR4 is also involved. In this study, they also reported that the ABC-transporter (MPN333), and F 0 F 1 ATP synthase subunit ε (MPN597) of M. pneumoniae are essential for the activation of the autophagy/TLR4-mediated pathway.
Interestingly, Bose et al. (2014) showed that CARDS toxin (MPN372) regulates NLRP3 inflammasome activity. CARDs toxin is a vacuolating cytotoxin produced by some Mycoplasma species, including M. pneumoniae. Its C-terminal region is responsible for its vacuolating activity (Kannan and Baseman, 2006;Kannan et al., 2014). Its N-terminal region shares sequence similarity with pertussis toxin and is essential for its ADP-ribosylating activity. In this study, they demonstrated that CARDS toxin activates inflammasomes through the ADP-ribosylation of NLRP3 and enhances the secretion of IL-1β.
Taken together, these findings suggest that inflammasomes play an important role in the inflammation induced by M. pneumoniae.

Cytadherence of M. pneumoniae and Inflammation
Cytadherence property is one of the unique characteristics of M. pneumoniae. Cytadherence in the respiratory tract, the initial event in M. pneumoniae infection, is mediated by P1 (MPN141) adhesin and other accessory proteins, such as P30 and HMW proteins (Krause and Balish, 2001;Balish and Krause, 2002;Miyata, 2008a,b). The relationship between cytoadherence and the induction of inflammatory responses was first reported in Yang et al. (2002). They demonstrated that protease treatment or anti-P1 antibody treatment decreases the induction of pro-inflammatory cytokines, including IL-1β. Hoek et al. (2005) reported that culturing M. pneumoniae in polypropylene bottles reduces the expression of P1 adhesin. Under these conditions, the induction of IL-4 from mast cells was significantly decreased. As described above, Shimizu et al. reported that M. pneumoniae induces the efflux of ATP from host cells, followed by the activation of inflammasomes and secretion of IL-1β. In this study, they also reported that cytadherence-deficient mutants lacking P90 and P40 (MPN142, 130 kDa precursor) or HMW1 and P30 (MPN447 and MPN453, respectively) fail to induce IL-1β through ATP efflux. Cytadherence was also associated with autophagy/TLR4mediated induction of inflammatory responses. Mutation in ABC-transporter (MPN333), and F 0 F 1 ATP synthase subunit ε (MPN597) failed to induce inflammatory responses, and these mutants showed a deficiency in cytadherence (Shimizu et al., 2014). Taken together, these findings indicate that cytadherence of M. pneumoniae is strongly associated with the induction of inflammatory responses.

CONCLUSION
In this review, the molecular mechanisms of inflammatory responses induced by M. pneumoniae were reviewed (Figure 2). The following four pathways are important for the induction of inflammatory responses in M. pneumoniae infection: 1) recognition of lipoprotein by TLR2, 2) autophagy-mediated signaling; 3) activation of inflammasomes, and 4) cytadherence property. Lipoproteins, which were the first immunostimulants discovered in Mycoplasma species, have been well studied. However, the structures of the lipoproteins in Mycoplasma species are identical to those of lipoproteins from other bacteria, including normal microflora. Therefore, lipoproteins alone are insufficient to explain the inflammatory responses induced by M. pneumoniae. M. pneumoniae also has the ability to induce inflammatory responses through a TLR2independent pathway. Autophagy and TLR4 are involved in this induction. Some pro-inflammatory cytokines, such as IL-1β and IL-18, are matured and released through inflammasome activation. Inflammasome activation is necessary to release these cytokines during M. pneumoniae infection. It is noteworthy that CARDS toxin enhances inflammasome activation. The distribution of CARDS toxin in Mycoplasma species is limited to a small number of Mycoplasma species. In addition, cytadherent property of M. pneumoniae is strongly associated with the autophagy/TLR4-and inflammasomemediated induction of inflammatory responses. Although some Mycoplasma species, such as M. genitalium and M. gallisepticum, have partially similar adhesin, cytadherence mediated by P1 adhesin is unique in M. pneumoniae. These characteristics may contribute to the greater ability of M. pneumoniae to induce inflammatory responses than nonpathogenic Mycoplasma species.
In addition to inflammation-inducing factors, M. pneumoniae shows cytotoxicity through CARDS toxin, nuclease, and hydrogen peroxide produced during glycerol metabolism. The symptoms of mycoplasmal pneumonia, such as fever and severe cough, are thought to appear as a result of a combination of inflammation and cytotoxicity induced by M. pneumoniae. Mycoplasmal pneumonia is still an important issue in the field of pediatric medicine. Although measures to prevent mycoplasmal pneumonia are desired worldwide, preventive measures, including vaccines, have not been developed. Therefore, the inflammation-inducing factors of M. pneumoniae described here may be suitable targets for the development of new preventive measures.

AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and approved it for publication.