Molecular studies on a Mip-like protein required in X-bacteria/Amoeba symbiosis and characterization of the myosin heavy chain gene in Amoeba proteus

Author

Sang Wook Oh

Date of Award

8-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Zoology

Major Professor

Kwang W. Jeon

Committee Members

John R. Kennedy, Bruce D. McKey, Ranjan Ganguly, Gary Stacey

Abstract

X-Bacteria, obligatory endosymbionts in the xD strain of Amoeba proteus, survive inside hosts and the symbiosomal membrane containing X-bacteria does not fuse with lysosomes, suggesting that certain component(s) from X-bacteria may contribute to this process. Meanwhile, the macrophage infectivity potentiator (Mip) as a surface polycationic protein is known to be a virulence factor, involved in the initial infection of Legionella as well as many pathogenic bacteria. Since recent studies on cell wall components in X-bacteria and nucleotide sequence of the groELx gene show that X-bacteria are closely related to Legionella species, the m/p-like gene was cloned and the function of Mip-like protein was investigated in X-bacteria/amoeba interaction.

A 30-kDa Mip-like protein (Mipx) in X-bacteria was detected by SDS-PAGE, 2-dimensional gel electrophoresis and immunoblotting using an antibody against Mip of Legionella pneumophila. Immunoblotting of X-bacterial proteins fractionated by detergents showed that Mipx is a membrane protein. Using conserved nucleotide sequences from Legionella mip genes as primers, a PCR product containing a m/p-like gene (mipx) was obtained from a genomic expression library of X-bacteria. The presence of mipx in X-bacterial genomic DNA was confirmed by Southern hybridization using a ³²P-labeled PCR fragment as a probe.

The mipx gene revealed that it contained one open reading frame (ORF) of 729 bp encoding Mipx protein of 243 amino acids. The Mipx protein was highly basic with a predicted pi value of 10.02 and its secondary structure corresponded very well to those of FKBP homologs. The mipx gene had a sequence identity of 79%, 74% and 61% with those of Legionella micdadei, L. pneumophila and Chlamydia trachomatis, respectively. Mipx contained amino acids corresponding to the peptidyl-prolyl cis-trans isomerase (PPIase) activity region at its C-terminus and six amino acids out of ten known to be involved in the interaction of huamn FKBP and FK506 were well conserved in Mipx.

The recombinant Mipx protein overproduced by E. co//exhibited PPIase enzymatic activity and its activity was inhibited by the immunosuppressant drug, FK506. For evaluating the role of Mipx in X-bacteria infection in amoebae, Xbacteria pretreated with FK506 were experimentally introduced into D amoebae. X-Bacteria’s infectivity in amoebae was lowered to one-third that of the control group, and the results indicated that Mipx may be involved in the initial infection of X-bacteria in amoebae. Since the recombinant Mipx-producing E. co//were being almost digested 3 days after infection into amoebae chased by mAb KJX5, the other factors as well as Mipx protein are thought to be involved in the survival of X-bacteria in amoebae.

Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNA gene encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, indicating that myosin is involved in the cellular organelle movement. The localization of myosin in xD amoebae on the symbiosome membrane was further confirmed by immunoelectron microscopy. The ORF of a cloned myosin cDNA gene contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While amino-acid sequence of the globular head region of amoeba myosin had a high degree of homology with those of myosin from various organisms, the tail region building an a-helical coiled-coil structure did not show a significant sequence similarity. Interestingly enough, the proline residue which is strictly conserved in all myosins and used as a landmark for the boundary between the head and tail domains was replaced with lysine in the amoeba myosin. There appeared to be at least three different isoforms of myosins in amoebae, with a high degree of homology in their globular head regions.

Recommended Citation

Oh, Sang Wook, "Molecular studies on a Mip-like protein required in X-bacteria/Amoeba symbiosis and characterization of the myosin heavy chain gene in Amoeba proteus. " PhD diss., University of Tennessee, 1998.
https://trace.tennessee.edu/utk_graddiss/9325