The fact that Mycoplasmas have a uniquely small genome and lack a cell wall would probably make us consider these pathogens to be harmless, but on the contrary, these pathogens have evolved to survive in changing environments and adapting to them even after being fastidious in their requirements. This essay discussed the pathogenesis of these organisms and their complex interactions with the host. These organisms have developed a parasitic mode of life so as to survive with such a small genome. They depend on the host for essential nutrients and compete with the host cells for the same. Even after depending on the host cell for its nutrients, these organisms are able to cause diseases in their hosts by interacting with the host cells and evading the immune system, as is concluded from this essay.
Mycoplasmas represent the smallest self-replicating organisms, in terms of cellular dimensions and genome size that are capable of a cell-free existence  .They are parasitic bacteria that lack a cell wall and are known to have the smallest genome size of 540-1300kb. Mycoplasmas belong to the class Mollicutes, separated from the other walled bacterial species. They were considered to be viruses when they were first discovered since they were able to pass through filters which could retain bacterial cells. However, unlike viruses, mycoplasmal cells are able to grow in cell-free media and contain RNA and DNA as their genetic material  . Due to their small genome these organisms were considered as the primitive organisms, however, there is solid genetic support from nucleic acid hybridization and sequencing studies which indicate that these organisms are derived from a branch of gram-positive bacteria  . Their survival with such a small genome was made possible by adopting a parasitic mode of life. It is hypothesised that the cell wall-encoding genes were lost during reductive evolution, where the size of their genome was greatly reduced, retaining those genes that were essential for life as seen in the present-day Mycoplasmas  .
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Besides having an exceptionally small genome, the Mycoplasmal DNA base-composition is unique. The A-T content of their DNA is extremely high, unlike in other organisms. Also, UGA is a stop codon is most organisms, but it encodes for the amino acid, tryptophan in most mycoplasmas  . Due to the limiting coding capacity of the mycoplasmal genome, they lack many metabolic pathways that are required for energy production and for the synthesis of many bacteria-specific enzymes. Cell wall absence in Mycoplasma enables them to have an elastic shape; they are bound by a single sterol-containing bilayered membraneâ€•a very significant feature to their existence.Â The inner leaflet of the bilayer is phospholipid; the outer leaflet is composed of lipoproteins  . As seen from table.1, these two features are unique to Mycoplasmas, helping the organism to survive within the host.
Feature in other bacteria
Advantage in Mycoplasma
Disadvantage in mycoplasma
Cell wall absent
Cell wall present
Elastic shape-filamentous, coccoidal, spherical, or granularâ€•depending on the species as well as the media that they are grown in 
Physical stability due to elastic shape
Susceptible to dehydration
Limited to parasitic mode of life, therefore follow a strict host and tissue specificity.
Sterol-containing cell membrane
Sterols absent in cell membrane
Cell membrane highly stable-steroids reduce membrane fluidity and flexibility 
Sterols contain lipoglycans that aid in movement by their adhesive properties with host cells8
Always on Time
Marked to Standard
Facilitate attachment to the surface receptors of animal cells.
Not synthesised by organism, therefore dependent on host tissue (acquired as cholesterol)8
Table.1 Unique features of mycoplasmas: The absence of a cell wall and a sterol containing cell membrane. These features have their own advantages and disadvantages, as listed above; however, they still help these organisms to survive within the hosts.
Mycoplasmas as a pathogen
Mycoplasmas are human urinogenital and respiratory tract parasites that adhere to epithelial cell surfaces via surface antigens present on their cell membrane, making adherence a major virulence factor  . Â In the absence of a cell wall, the majority of Mycoplasmal surface-variable antigens are lipoproteins. Some surface proteins undergo spontaneous antigenic variation, as discussed further through the essay. They attach themselves to specific receptors on the host cells, producing no symptoms, hence their infection can go undetected  . Due to the lack of a cell wall, it is very difficult to isolate and confirm the presence of Mycoplasma in the laboratory. Only after other pathogens are eliminated as the cause of disease, is Mycoplasma considered as a cause and treated directly. Î²-lactams which prevent cell wall synthesis are ineffective on Mycoplasmas; hence macrolides and tetracyclines are used for treatment2. Mycoplasmas are usually detected via direct DNA stains, ELISA and autoradiography, but PCR tests are known to be most accurate.
There are over 200 species of Mycoplasmas, widely distributed in humans, vertebrate animals and plants1. Most are innocuous and harmless, living as parasites primarily in the respiratory and urinogenital tracts of animals and humans. However, these innocuous species may aggravate secondary infections, if triggered. Table2 summarises few pathogenic mycoplasma species and the range of diseases caused by them.
Primary site of colonization
contagious bovine pleuropneumonia
Gulf war syndrome, autoimmune diseases
Postpartum fever, localized abscesses in immunosuppressed patients.
Urinogenital and Respiratory tract
Aids in deterioration of immune system in HIV
Atypical pneumonia, asthma, CNS disorders
Table.2: Pathogenic Mycoplasma species that infect different hosts3
Mycoplasma pneumoniae accounts for 10% of all pneumonia cases and is known for stimulating the production of autoantibodies, leading to secondary manifestations. Fig.1 shows a chest radiograph of M.pneumoniae infected lungs. In this essay I discussed the strategies employed by Mycoplasma while interacting with host eukaryotic cells and with the cells of the immune system, concentrating on Mycoplasma pneumonia, an extracellular parasite in humans.
Fig.1. Chest radiograph of an atypical pneumonia patient with bilateral patchy alveolar opacities of the lower lobes.Â Taken from: MarrieÂ TJ. Communityâ€acquired pneumonia.Â Â Clin Infect Dis. 1994; 18: p.501
Mycoplasmas' relationship with the host
Even though Mycoplasmas are known to occur innocuously in the respiratory tract and urinogenital tract of humans, they are known to cause or be a significant co-factor to many other diseases like Crohn's disease, nongonoccal urethritis, asthma and rheumatoid arthritis. Many Mycoplasmal species are innocuously present extracellularly; however, some species can invade the host cells and reside intracellularly, causing diseases.
Mycoplasmal attachment to their target cells is a fundamental step in tissue colonization and disease onset  . M.pneumoniae and M.genetalium have developed a complex terminal structure called attachment organelle which assists in polar localization of surface proteins called adhesins, allowing adherence via interactions with receptors on the host cell surface  . Adhesins on the attachment organelle bind the parasite to sialoglycolipid receptors at the base of the cilia on the epithelial cell surface with the help of sialic acid residues that are present on the respiratory epithelial cells of the host. A 170-kilodalton(kDa) protein, called P1 is the major adhesin while a 30-kDa protein called P30, a transmembrane protein, is also densely clustered at the M.pneumoniae attachment organelle, aiding in the cytadherence process. The presence of multiple adhesins suggests that there are multiple receptors for M.pneumoniae on the host cell surface which allows the organism to adhere  . Fig.2 shows a schematic diagram of M.pneumoniae adhering to the ciliary epithelium.
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Fig. 2 Schematic presentation of aÂ M pneumoniaeÂ organism attaching to the surface of the ciliary tracheal epithelium via the attachment organelle, as seen by electron microscopy of a thin section taken from:
Baron, Samuel 1996. "Mycoplasmas."Â Medical Microbiology. 4th ed. New York: Churchill Livingstone
The P1 adhesin is encoded by the P1 gene which is part of a three-gene operon. This operon has 3 open-reading frames: ORF-4, P1, ORF-6. Sequence and size analysis of these genes indicated that they encode a 28kDa, 170kDa and a 130kDa protein respectively  . While testing for the protein encoded by ORF-6, it was determined that two proteins of 40kDa(P40) and 90kDa(P90) were formed by proteolytic cleavage  . Since genes on the same operon usually carry out the same function, P40 and P90 are called accessory proteins, found along with P1-proteins and help in M.pneumoniae adhesion14.
Besides P40 and P90, another group of 3 accessory proteins, HMW1, HMW2 and HMW3 are involved in cytadherence. Those organisms that lack P40 or P90 proteins tend to have the P1-protein scattered on their membrane;P1 fails to cluster at the tip of the attachment organelle13. Therefore, absence of HMW1-HMW3 proteins leads to the loss of the characteristic truncated appearance of the attachment organelle that is usually seen in the wildtype organisms. These alterations suggest that accessory proteins are required for maintaining the proper shape of the attachment organelle and HMW3 plays an important role in maintaining the architecture and stability of the organelle14. HMW2 is known to play a regulatory role; loss of HMW2 protein leads to reduced levels of other accessory proteins, hence directly affecting cytadherence. Accessory proteins,therefore, do not participate in receptor binding of the pathogen to the host cell but play a vital role in the lateral movement and maintenance of adhesins at the tip of the attachment organelle13. Fig.3 shows a schematic diagram of all the cytadhering proteins in the attachment organelle.
Fig.3 Schematic diagram of the attachment organelle and the location of the major cytadherence and accessory proteins inÂ M.Â pneumoniae .The P1 and P30 adhesin are the major cytadhesins clustered at the tip of the organelle. HMW3 protein helps in maintaining the architecture of the organelle. Taken from:
Razin, Shmuel, and Richard Herrmann. "Cytadherence and the Cytoskeleton."Â Molecular Biology and Pathogenicity of Mycoplasmas. New York: Kluwer Academic/Plenum, 2002. 491-518. Print.
Adherence is followed by severe oxidative damage of host epithelial cells by hydrogen peroxide and superoxides produced by the adhered M.pneumoniae organisms12. These powerful oxidants induce oxidative stress, leading to host membrane damage. It is essential for the pathogen to adhere very closely to the host cell in order to maintain the high concentrations of the peroxide required to have a toxic effect. The superoxide inhibits host cell catalase, increasing the accumulation of hydrogen peroxide at the contact site12, as seen in fig.4.
Fig.4 Proposed mechanism of oxidative damage to host cells by adheringÂ M pneumoniae. The high concentration of peroxide in and around the cell causes damage to the cells, hence causing an inflammatory response. Taken from Baron, Samuel (1996). "Mycoplasmas."Â Medical Microbiology. 4th ed. New York: Churchill Livingstone
Thus, binding of Mycoplasmas to the eukaryotic cells makes the cell more permeable to all the toxic substances these pathogens produce6. Attachment of M.pneumoniae to the epithelial cells leads to the inhibition of the "beating" of ciliated cells, inactivating a vital defence of the upper respiratory tract.
Interactions of pathogens with the cells of the immune system
Mycoplasmas are able to exert a range of specific and non-specific immune reactions after attachment to host cells. Pathogen adherence to the host cell induces a specific immune response by generating serum and antibodies; this indicates that Mycoplasmas stimulate the production of B-lymphocytes.The first response occurs after an initial infection with M.pneumoniae with the production of M.pneumoniae-specific IgM (immunoglobulin-M) antibodies followed by predominance of IgG and IgA-antibodies  . Once the specific antibody has opsonised the pathogen, phagocytosis by leukocytes and macrophages occurs.
Leukocyte stimulation occurs by a non-specific immunomodulatory effect via a cascade of reactions, originally triggered by the pathogen lipoproteins. Lipoproteins are immunomodulins, i.e. they induce cytokine synthesis. Cytokine synthesis comprises a cascade of reactions starting with the Toll-like receptors(TLRs)  , a 10-protien family which recognize and bind to structurally conserved molecules associated with microbes. TLR2, known to recognize lipoglycans, recognizes a 21kDa dipalmitoylated lipoprotein subunit-b of F0F1-type ATPase  present in the M.pneumoniae membrane and activates Nuclear Factor-kB(NF-kB) via signal molecules13. NF-kB activates the transcription of a large number of genes encoding proteins involved in immunological processes, specifically leading to monocyte and macrophage stimulation, which then secrete proinflammatory cytokines like Tumour-Necrosis Factor(TNF)-Î±, Interleukin(IL)-1 and IL-612,each performing different functions, as seen in table.3. Hence, cytokine production can either have a positive effect by minimizing disease by enhancing host defence system, or have a negative effect by worsening damage and inflammation, causing immunologic hypersensitivity. There is evidence from human studies that suggests that severity of the disease is directly linked with the extent of cytokine production and leukocyte stimulation. Thus, a high cell-mediated immune response results in severe clinical illness and pulmonary injury.
Function of cytokine in the immune response
Promote B&T-lymphocyte proliferation into effector cells
Principle mediator of inflammation, fever and release of acute phase proteins
UpregulateÂ the cytocidal activity of macrophages
Stimulation of leukocytes and the expression of major histocompatibility complex(MHC) class-I and class-II antigens on macrophages, enhancing antigenic presentation by APCs.
Cofactor in B-cell differentiation and maturation
Enhance cytotoxic activity of NK cells
Table3. Cytokine induction in human hosts by M. pneumoniae during infection
Along with the induction of cells of the host immune system and cytokine production, M. pneumoniae infection leads to the development of autoantibodies. Autoantibody production is suggested to be an effect of extensive sequence homology between P1 adhesin molecule of the pathogen and host cell-membrane proteins, referred to as molecular mimicry. It is proposed that M.pneumoniae stimulate autoreactive T-cells that are directed against the pathogenic antigens, but due to extensive sequence homology, these antibodies also attack host cells while destroying M.pneumoniae  . Among these are antibodies to the brain, IgM-class cold agglutinins, antibodies to lung tissues and smooth muscle  .
Cold agglutinins, for example, are IgM-autoantibodies that react with surface antigens of erythrocytes. They are specific to the I-antigen on erythrocytes' membrane surface and are produced in 50% of atypical pneumonia cases. It is proposed that M.pneumoniae adheres to erythrocyte membranes and hydrogen peroxide produced by M.pneumoniae alters the I-antigen, making it more antigenic, hence, I-antigen antibodies are produced  . Similarly, autoantibodies to glycolipids in the brain tissue are developed as cross-reaction of the M.pneumoniae-specific antibodies. M.pneumoniae infection precedes 5% of Guillain-Barré syndrome, where autoantibodies against galactocerebroside, a neural myelin antigen are produced. M.pneumoniae P1-adhesin also has sequence homologies with human CD4 and MHC-class-II proteins which trigger immunosuppression.
The sequence homology between M.pneumoniae and host cells triggers the development of autoantibodies and serves as a mechanism to evade the host defence system since this enables the pathogens to go unnoticed by the immune cells by making themselves look like host cells.
Evasion of host immune responses by Mycoplasma pneumoniae
The host immune system responds effectively after M.pneumoniae adheres to the epithelial cells with the chemotactic migration of macrophages and leucocytes to the infected site. In order to survive, these pathogens have evolved molecular mechanisms that enable them to evade the host immune response, enabling them to survive in the host. Antigenic variation is one of the major mechanisms evolved by M.pneumoniae in order to evade immune responses and adapt to the rapidly changing microenvironment within the host.
Antigenic variation refers to the ability of M.pneumoniae to alter the antigens on its membrane surface-proteins, producing multiple forms of morphology. In M.pneumoniae, distinct cell populations are generated, each having a distinct set of antigens on its surface that is able to survive specific host responses which eliminate another population of the same organism. This is a major cause of reinfection in patients that have been infected with this pathogen before.
The genetics of M.pneumoniae gives a significant explanation for antigenic variation in the organism. The gene encoding for the P1 protein was studied extensively, which revealed that there is only one copy of the entire P1 gene; however, two-thirds of the gene sequences were present in other parts of the genome as multiple copies while some regions occur as closely homologous, but not identical, multiple copies. These homologous and identical sequences provide variability while regulating the structural and functional properties of adhesins. Hence, high-frequency surface-adhesin variation by different rearrangements and recombinations of repeated elements and with regions from the P1-operon allow diversity and altered specificities in these organisms.
Genetic systems that enable antigenic variation in M.pneumoniae and other Mycoplasmas
As mentioned above, antigenic variation plays a key role in pathogen persistence within the host by aiding immune response evasion, hence establishing a prolonged infection. Although Mycoplasmas have an extremely small genome, the gene numbers involved in varying the surface antigenic properties are large. This process occurs in Mycoplasmas via random variation of genes encoding membrane proteins that are present in multiple copies in the mycoplasmal genome and are displayed on the surface in diverse recombinations  .
To maintain surface variation, Mycoplasmas use many structural genes with similar characteristics, organized as gene families. This allows generation of different antigen combinations, with each gene having the ability to switch on/off and being able to generate distinct size variants.
Antigenic variation can occur via two known mechanisms:
1.Variation via homopolymeric repeats: Some Mycoplasmas have small repeats of reiterated bases within gene sequences, quite often in which, insertion or deletion of nucleotides can occur. This can "switch on" or "switch off" a gene encoding a protein by generating frameshift mutations, causing premature termination of gene translation, resulting inÂ different size variants of the protein. Insertion/deletion of nucleotides occurs by a process called slipped-strand mispairing, in which, transient misalignment of the mother-daughter strand occurs during replication  . The location of these repeats can affect transcription or translation, hence regulating the genes' expression level.
This is observed in M.hyorhinis, where multiple repeats of adenine-residue strings are present in the conserved promoter sequences of the vlp-genes, encoding a set of variable lipoproteins  . Nucleotide insertion in these repeats "switch off" the gene by increasing the length of the promoter, interfering with RNA polymerase positioning, hence preventing the transcription of the gene  .
Repeats of 7-adenine residues were found in the cytadhesin P1-gene of M.pneumoniae which underwent spontaneous mutations. Insertion and deletion of single nucleotides in these repeats resulted in the formation of reversible cytadherence-negative/cytadherence-positive mutants resulting from a frameshift mutation that generated a termination codon due to which the P1-gene couldn't be translated completely.
2.Variation via chromosomal rearrangements: This mechanism involves spontaneous DNA rearrangements by inversions, duplications or translocations of tandem blocks of DNA during replication and is used by Mycoplasmas to regulate variable surface-antigens. Rearrangement is aided by homologous recombination, dependent on the RecA function. RecA enhances the annealing of single-stranded DNA to a complementary sequence in the double-stranded DNA, replacing one of the old strands. For homologous recombination, extensive homology between gene sequences is required, therefore gene families are common targets, leading to the production of numerous phenotypic variants.
This is observed in the vsa-gene family encoding V-1 antigens of M.pulmonis. Sequence analysis of vsa-genes from two M.pulmonis organisms of different phenotypes(expressing, non-expressing) suggested that a chromosomal inversion caused these two genotypes. The 5'-promotor present in the expressed gene was missing in the silent gene; hence, re-assortment between the 3'-end of a silent gene and the 5'-end of the expressed gene regulates gene expression  .
The ability of these pathogens to maintain an antigenically versatile surface-membrane enables them to persist in the host organism by adapting to the changing microenvironment  , and evading the host immune response.
After discussing the complex interactions of Mycoplasmas with their hosts, it is evident that these pathogens not only have the ability to induce an immune response, but also evade the immune response very effectively. This enables them to persist in the host, causing prolonged infection. Although these organisms have the smallest genome of only 540 to 1300 kb, an infection caused by them can also lead to the development of autoantibodies within the host, hence causing secondary manifestations of mycoplasmal diseases. Even though not all infections caused my Mycoplasmas have a high mortality rate, these pathogens cause communicable infections and have a wide range of hosts including humans, animals and plants. These organisms have evolved over the last few decades to become more pathogenic than they were; this implies that they will evolve further to become more dangerous and more pathogenic in the future. Hence detailed study about the pathogenesis and interactions of this pathogen with the host holds great importance for future generations.
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