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Keywords:

  • Mycoplasma pneumoniae;
  • Organelle biogenesis;
  • Gliding motility;
  • Cytadherence;
  • Cell division

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity. Despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle that functions in cytadherence, gliding motility, and cell division. The attachment organelle is a membrane-bound extension of the cell and is characterized by an electron-dense core that is part of the mycoplasma cytoskeleton, defined here for working purposes as the protein fraction that remains after extraction with the detergent Triton X-100. This review focuses on the architecture and assembly of the terminal organelle of M. pneumoniae. Characterizing the downstream consequences of defects involving attachment organelle components has made it possible to begin to elucidate the probable sequence of certain events in the biogenesis of this structure.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity [1]. The finding that mycoplasmas lack the genes for the major pathways for synthesis of cell building blocks comes as no surprise to those who attempt to cultivate these fastidious bacteria. However, the lack of alternative sigma factors, two-component systems, or other means of gene regulation common to prototypical walled bacteria is almost as striking as their absence of a cell wall [1]. Yet despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle [2]. This review focuses on the role of certain proteins in the architecture and function of this structure, including new information from mutant analysis regarding the likely sequence of events in the assembly of a fully functional terminal organelle.

2Mycoplasma pneumoniae

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

M. pneumoniae causes bronchitis and community-acquired, atypical or ‘walking’ pneumonia in humans [3]. Long considered a pathogen primarily of children and young adults, in whom M. pneumoniae is the leading cause of pneumonia, this species is now more widely recognized as a health threat to people of all ages. Symptoms may persist for weeks and are generally nondescript and flu-like in nature, typically with a low-grade fever and a chronic, non-productive cough. Extra-pulmonary sequelae are not uncommon, but fatal infections are rare.

M. pneumoniae colonizes the bronchial passages, localizing to the base of the cilia where it interacts directly with the host cell surface [1]. Evidence is mounting for host cell invasion by several mycoplasma species previously considered to be strictly surface parasites, which might explain how M. pneumoniae establishes carrier states or could account for the difficulty in clearing infections effectively with antibiotic treatment [3,4]. However, invasion by M. pneumoniae has been demonstrated to date only with host cells in tissue culture and not with differentiated host tissue in vitro or in vivo and therefore should be interpreted cautiously.

M. pneumoniae colonization results from the interaction between adhesin proteins on the mycoplasma surface and sulfated glycolipid or sialoglycoprotein molecules on the host respiratory epithelium [5]. The mycoplasmas are often seen with the differentiated terminal organelle oriented toward, and directly associated with, the host cell surface, hence the common designation ‘attachment organelle’ for this structure. The attachment organelle is a membrane-bound extension of the bacterial cell rather than a surface appendage. It is characterized by an electron-dense core oriented lengthwise and enlarging to form a terminal button which interfaces with the inner surface of the mycoplasma membrane at the tip of the cell (reviewed in [2]). The electron-dense core is part of the cytoskeleton, which is visualized through electron microscopy as a filamentous network throughout the mycoplasma cell and defined for working purposes as the protein fraction that remains after extraction of M. pneumoniae cells with the non-ionic detergent Triton X-100 [2,6,7]. Several other mycoplasma species have terminal organelles but which differ in shape and appearance from that of M. pneumoniae[8].

The term ‘attachment organelle’ is somewhat misleading, as this structure is thought to be multifunctional at some level. Thus, for example, the terminal organelle is the leading end as M. pneumoniae cells move by gliding motility. Mycoplasma gliding can be observed by microcinematography and requires a solid–liquid interface [9], but the underlying mechanism is not understood, and annotation of the M. pneumoniae genome reveals no ORFs predicted to be involved in motility or chemotaxis based on similarity to genes of known function [10]. The culture of other gliding bacteria on soft agar results in satellite growth, and we have likewise recently developed a means to visualize satellite growth with M. pneumoniae (Fig. 1), a capability that should allow identification of non-motile mutants and association of specific genes and gene products with gliding motility. Such studies should complement the analysis of gliding motility in Mycoplasma mobile, which likewise has a differentiated terminal organelle but for which no genetic system has been reported [11].

image

Figure 1. Satellite colony formation due to gliding motility by wild-type M. pneumoniae cultured on soft agar with a solid–liquid interface. Photo courtesy of J. Jordan.

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The M. pneumoniae terminal organelle is also thought to play an important part in cell division [8]. It has not been possible to definitively establish the sequence of events in mycoplasma cell growth and development due to their small size and poor optical qualities. However, cell images by scanning and transmission electron microscopy suggest that duplication of the terminal organelle precedes cell division (Fig. 2). Whether the existing tip divides or a new tip forms adjacent to the first is not clear; nevertheless, one tip structure then moves along the mycoplasma cell to the opposite pole, and this is followed by cytokinesis. This scenario raises the possibility that the terminal organelle may also function in the segregation of daughter chromosomes. Recent findings by Seto et al. [12] clearly establish this temporal relationship between chromosome replication and the formation and movement of the second tip structure. One might therefore predict that mutations that result in loss of cytadherence could also confer a developmental defect. A number of mycoplasma non-cytadhering mutants have been isolated and characterized, and as a result a significant group of proteins has been functionally associated with cytadherence (Table 1; reviewed in [13]). The detailed analysis of these mutants is beginning to elucidate how these proteins may actually function in cell development and the assembly of the terminal organelle.

image

Figure 2. Probable sequence of events in M. pneumoniae cell division. Duplication of the terminal organelle is followed by movement of the original or nascent organelle to the opposite end of the cell and cytokinesis.

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Table 1.  Summary of protein differences in certain non-cytadhering mutants of M. pneumoniae
ProteinProtein level in M. pneumoniae strain
 wild-type M129mutant I-2amutant II-3amutant M6bmutant IIIamutant IVa
  1. aFrom [16].

  2. bFrom [17].

  3. cArbitrary units standardized to wild-type M. pneumoniae.

  4. dProtein P30 is truncated at the C-terminus in this mutant.

HMW11c0.21011
HMW21010.511
HMW310.210.511
P3010.700.5d11
P2810.210.511
P1111110
P6510.10.20.111
B111100

3M. pneumoniae cytadherence proteins

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

3.1Protein P1

The membrane protein P1 is thought to have a major receptor-binding role in M. pneumoniae[1,2,5]. Considerable information exists regarding which domains of P1 function in receptor binding and which domains are targeted by circulating and secreted antibodies in human infections [14], and the implications thereof on immune evasion, but this aspect of M. pneumoniae molecular biology and the underlying biological mimicry is beyond the scope of this review. P1 is localized primarily to the attachment organelle in wild-type mycoplasmas but is also found scattered elsewhere on the mycoplasma surface. Synthesized in precursor form, a processing event is required to yield mature P1. The leader peptide [15] consists of a typical, positively charged N-terminus followed by a hydrophobic core that ends with alanine residues in what would ordinarily constitute the −3 and −1 positions of a signal peptidase I cleavage site (Fig. 3). Based on the N-terminal amino acid sequence of mature P1, however, cleavage actually occurs at a position 36 residues downstream of this putative signal peptidase I site in order to generate mature P1. Significantly, M. pneumoniae appears to lack the gene for signal peptidase I [10], although this does not rule out the possibility of a two-step processing event. Alternatively, processing of P1 may require only the one cleavage event at the downstream site. The significance of these findings to P1 maturation and function is considered in more detail below.

image

Figure 3. Leader peptide of protein P1. Single-letter amino acid designations are used. The underlined residues correspond to the putative −3 to −1 region at the C-terminal end of the hydrophobic core corresponding to a likely signal peptidase I cleavage site.

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3.2Cytadherence-accessory proteins

P1 alone is not sufficient for mycoplasma attachment to host cells. A number of spontaneously arising mutants have been isolated by screening individual colonies for hemadsorption, a convenient indicator for adherence to respiratory epithelium [16,17]. All but one of these mutants possess P1, and for most of those, P1 fails to localize to the attachment organelle but is widely scattered elsewhere on the mycoplasma surface [18–20]. This observation led to the conclusion that proper trafficking of P1 requires cytadherence-accessory proteins, and more recent analysis has shed additional light on the defect in P1 localization [21]. In wild-type mycoplasmas the P1 precursor was detectable in pulse-chase labeling experiments, and processing to yield mature P1 was essentially complete within 1 h. However, the processing of pre-P1 occurred considerably more slowly in the cytadherence mutants, where the P1 precursor was detectable at substantial levels for several hours post-labeling. It is not clear whether the failure of P1 to localize correctly is due entirely to this defect in processing, or perhaps vice versa. Nevertheless, if one assumes that the final folding of P1 takes place only once it reaches the proper subcellular compartment, perhaps the long leader peptide is necessary to stabilize pre-P1 in an intermediate conformation. The inability to form a fully developed terminal organelle might therefore delay the localization of P1 to the proper subcellular compartment, thereby restricting the final steps in processing and folding.

The number of proteins associated with cytadherence is surprisingly large (Table 1), raising the possibility that most are not directly involved with receptor binding but might be structural elements of the attachment organelle [18]. These cytadherence-associated proteins are largely components of the cytoskeleton, based on their partitioning in the Triton X-100-insoluble fraction [2,22]. Furthermore, loss of these cytadherence-associated proteins is accompanied by dramatic changes in cell morphology affecting the presence and appearance of the terminal organelle [2,12,19].

One such protein is HMW1, which garnered much initial attention largely out of convenience in that it is both highly immunogenic and easily discernible by SDS–PAGE. Analysis by immunoelectron microscopy established a bipolar localization for HMW1 to both the leading and trailing filaments of the mycoplasma cell [23]. More recent examination by immunofluorescence microscopy confirmed a polar location for HMW1 but failed to detect HMW1 along the trailing filaments, perhaps reflecting a difference in the sensitivity of the two techniques [12]. Loss of HMW1 results in a striking change in morphology, with the attachment organelle no longer distinguishable by scanning electron microscopy [19]. Recombinant HMW1 restores a more wild-type morphology, but a slight C-terminal truncation renders this recombinant HMW1 non-functional. Significantly, the C-terminus of HMW1 seems to be required both for full function and for proteolytic targeting in non-cytadhering mutants that lack HMW2 (Table 1 and discussed below [19,21]). HMW1 contains an unusual central domain that is dominated by repeating acidic and proline-rich motifs [24]. This structural feature is shared with cytadherence-associated proteins HMW3 and P65 (Table 1) as well as protein P200 [22,25], and while its significance in the function(s) of these proteins is not clear, all four proteins are components of the detergent-insoluble, cytoskeletal fraction [22,25]. HMW1 localizes to the mycoplasma cell surface, but how it crosses the cell membrane is not known, as HMW1 has neither a leader peptide sequence nor a significantly hydrophobic domain.

Protein P30 is likewise essential to cytadherence, and P30-specific antibodies block attachment to host cells [26], suggesting a direct role in receptor binding. P30 is a transmembrane protein oriented with its C-terminus to the cell exterior and having what is probably a small intracellular domain [27]. The cell-surface domain of P1 is dominated by proline-rich repeats that are essential for normal P30 function [17,27]. Particularly striking is the cascade of effects associated with loss or truncation of P30, including loss of cytadherence and gliding motility, a dramatic, branched morphology, and a diffuse nucleoid [12,20]. Similar changes in morphology and appearance of the nucleoid were described recently in other M. pneumoniae cytadherence mutants [12], consistent with a developmental defect [20]. Nevertheless, normal localization of P1 [12,20], HMW1, HMW3, B and C [12] was observed in the mutant having a truncated P30, the possible significance of which is addressed below.

A striking cascade of effects is likewise associated with a frameshift in the hmw2 gene in cytadherence mutant I-2, where loss of HMW2 is accompanied by reduced levels of HMW1, HMW3, P65, and P30 [16,28,29]. HMW1 and HMW3 are actually synthesized at wild-type levels in this mutant but are subject to accelerated proteolysis in the absence of HMW2 [21], and recent studies indicate that the same is true for P65 (manuscript in preparation). More recent analysis of HMW1 by pulse-chase studies [30] clearly demonstrates that newly synthesized HMW1 begins in the cytoplasmic (Triton X-100-soluble) fraction, rapidly shifts to the cytoskeleton fraction, and eventually localizes to the surface of wild-type mycoplasma cells. In the absence of HMW2 the newly synthesized HMW1 is not processed efficiently to the cell surface, leading to accelerated turnover in the cytoplasmic pool.

Synthesis of HMW2 at normal levels from a recombinant wild-type hmw2 allele introduced by transposon delivery fully restores cytadherence and wild-type levels of HMW1, HMW3, and P65 [29]. However, low-level production of HMW2 in some transformants only partially restored a normal phenotype, with the levels of P65 and HMW1 correlating directly with the amount of recombinant HMW2 produced. Thus, proper stoichiometry of these cytadherence-associated proteins appears to be particularly important.

HMW2 is predicted to have a periodicity that is highly characteristic of a coiled-coil conformation, which is typical of filamentous domains of known cytoskeletal proteins [31]. However, the apparent disruption of the coiled-coil motifs in several places suggests a conformation that is more like a flexible chain than a rigid rod. Five leucine zipper motifs are dispersed in the middle of HMW2, providing additional potential for dimerization interactions. The subcellular location of HMW2 in M. pneumoniae has been an enigma for some time. HMW2 is a major cell component and therefore available in substantial quantities (by mycoplasma standards) but is poorly immunogenic. Antibodies produced against fusion proteins containing N- and C-terminal sequences of HMW2 react with denatured HMW2 in Western immunoblots [31] but not in various subcellular fractions or in thin sections. As an alternative approach to localize HMW2 in M. pneumoniae, we engineered a translational sandwich fusion of HMW2 and green fluorescent protein (GFP). The fusion protein was stable and functional in an hmw2 mutant and was localized by fluorescence microscopy to the attachment organelle (manuscript in preparation). Immunoelectron microscopy studies are under way using antibodies to the GFP domain to localize this fusion protein more precisely in the mycoplasma cell. Based upon that information it should be possible to conjecture how HMW2 may function to effect efficient delivery of HMW1 to the mycoplasma surface. In the meantime, these findings clearly demonstrate that GFP can be a powerful tool even in cells as small as mycoplasmas.

M. pneumoniae gene MP012 ([32]; formerly orf6 or E07_orf1218 in the P1 operon [10]) encodes a 130-kDa polypeptide that is subsequently processed to yield proteins B and C (also known as P90 and P40, respectively; reviewed in [2]). Translation of the 130-kDa polypeptide appears to be coupled to the translation of P1, the gene for which immediately precedes orf6. Loss of B and C results in the inability to cytadhere, failure to localize P1 to the terminal organelle, and an atypical appearance to the tip structure [18]. Seto et al., however, report that P1, as well as HMW1, HMW3, and P30, all localize properly in an independently isolated mutant lacking proteins B and C [12]. The reason for the discrepancy regarding P1 localization is not clear but may reflect an undefined difference between the two mutants. Nevertheless, chemical cross-linking analysis has established that B, C, and P1 exist in very close proximity on the mycoplasma surface (reviewed in [2]), a finding now confirmed and extended to include HMW1 and P65 [33]. Finally, the loss of B and C appears to have no effect on the stability of other cytadherence-associated proteins, and the stability of B and C is unaffected by the loss of P30, HMW1, or HMW2 (Table 1; unpublished data).

4Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

The data generated from the detailed analysis of cytadherence mutants leads us to several important conclusions. Many of the cytadherence-associated proteins identified from these studies are components of the attachment organelle and are required for the proper assembly of this structure. Proper stoichiometry is clearly critical, and based upon the cascade of events that accompanies the loss of certain proteins, the likely sequence of events in the assembly process begins to emerge. Thus, we predict that the incorporation of organelle components occurs by at least two independent pathways that converge prior to P1 localization (Fig. 4). The loss of HMW1–HMW3, P30, or P65 has no bearing on the levels of proteins B and C, or vice versa. Nevertheless, all are required for proper processing and localization of P1. Furthermore, HMW2 and HMW1 appear to be critical early components, as their absence has a destabilizing effect on ‘downstream’ proteins. Less widespread consequences on the stability of other attachment organelle proteins or the localization of P1 to the tip structure are associated with the loss of P30 [20], and recent studies suggest that the same is true for HMW3 [34], suggesting that these proteins are incorporated later in the assembly process. In the event that a key protein is absent or present at reduced levels, downstream proteins that rely on that component to be localized and stabilized efficiently in the assembly process are subsequently turned over at an accelerated rate, probably by housekeeping proteolytic activity. Finally, B and C are coupled translationally to P1, and may very well precede P1 in the assembly process. Current data do not allow us to make that determination yet, however. This model thus expands upon the assembly scheme recently proposed by Seto et al. [12].

image

Figure 4. Predicted sequence for incorporation of cytadherence-accessory proteins into a nascent attachment organelle.

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A paradox exists then regarding how a ‘simple’ microorganism such as M. pneumoniae coordinates the assembly of the terminal organelle with cell division. In the absence of typical transcriptional regulators, how is the synthesis of component proteins of the terminal organelle controlled? Perhaps there is a trade-off for evolution via chromosome reduction with respect to maintaining the efficient utilization of carbon and energy, i.e., there is no means to regulate other than to degrade proteins unable to be utilized. The more likely expectation is that regulation occurs by novel means that are both energetically favorable and consistent with a limited regulatory repertoire. Given the importance of mycoplasmas to public health, the answer will be significant from both medical and biological perspectives.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References

Portions of this work were supported by Public Health Service Grants AI23362 and AI33396 from the National Institute of Allergy and Infectious Diseases to D.C.K. The authors regret that many fine studies were not cited specifically in this review due to the space restrictions. More complete reference listings can be found elsewhere [1,2,13,15].

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Mycoplasma pneumoniae
  5. 3M. pneumoniae cytadherence proteins
  6. 4Conclusions
  7. Acknowledgements
  8. References
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