Journal: Emerging Microbes & Infections

Article Title: Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis

doi: 10.1080/22221751.2024.2373317

Figure Lengend Snippet: Experimentally induced keratitis in NZW rabbits after intrastromal injection of T. pulmonis -PW1004. (a) Gross appearance of the rabbit eyes after Tsukamurella infection. (b) Mean bacterial load recovered from the cornea of rabbits infected with T. pulmonis -PW1004 and those of control rabbits at 24 h PI. Error bars indicated mean CFU/cornea ± SEM of three independent experiments. (c) Immunohistochemical staining of corneal sections using mouse anti- T. pulmonis -PW1004 serum. The boxed area is further enlarged and shown in the right-hand panel of the corresponding image. Strong positive staining in brown colour against T. pulmonis could be detected in corneal sections from rabbits infected with T. pulmonis -PW1004 (top) but not from the mock-infected control rabbits (bottom). The middle panel shows corneal sections from the infected rabbits stained with pre-immune control serum; corneal sections of infected rabbits showing large amount of inflammatory cell infiltration with haematoxylin counterstain (top and middle). (d) MPO activity (U/mg) of the corneal tissues harvested from rabbits. Error bars indicate mean ± SEM of three independent experiments.

Article Snippet: Although over 21 “ Tsukamurella species” have been described, using in silico genome-to-genome comparison as the standard of classification, only 16 Tsukamurella species should be included in this genus according to the current state of the taxonomy at the time of writing ( https://lpsn.dsmz.de/genus/tsukamurella ).

Techniques: Injection, Infection, Control, Immunohistochemical staining, Staining, Activity Assay

Journal: Emerging Microbes & Infections

Article Title: Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis

doi: 10.1080/22221751.2024.2373317

Figure Lengend Snippet: Characterization of the 3 tmyt homologues in Tsukamurella. (a) Locations of the tmytA, tmytB, and tmytC gene in the genome of T. pulmonis -PW1004 are indicated. Alignment of the tmyt homologues identified in T. pulmonis -PW1004, T. tyrosinosolvens -PW899, M. tuberculosis (GenBank accession numbers NP_218321, NP_216402, YP_177694 and YP_178017) and C. glutamicum (GenBank accession numbers AAAP23202-AAAP232007). The catalytic triad formed by functional residues Ser125, Asp/Glu229, and His261, which are important for mycolyltransferase activity, are indicated by black, grey, and dark grey boxes, respectively. (b) Biofilm formed by the PW1004-WT and its derivative mutants when they were cultured under static conditions for 3 days. With the exception of the PW1004Δ tmytC , dense and confluent biofilm was formed as a floating pellicle at the air-liquid interface in PW1004Δ tmytA , PW1004Δ tmytB , PW1004-WT, and tmytC complemented mutant (PW1004Δ tmytC /p tmytC ). (c) Quantitation of the biofilm formed by the PW1004-WT and its derivative mutants using crystal violet staining method. The amount of biofilms was significantly reduced in PW1004Δ tmytC compared to the PW1004-WT ( P < 0.01) and complemented mutant ( P < 0.05). (d) SEM and (e) confocal microscopy analyses of Tsukamurella biofilms cultured under static conditions for 3 days. Representative SEM micrographs of the biofilm formed by the PW1004Δ tmytC was flattened and less structured compared to those formed by the PW1004-WT and complemented mutant. Likewise, biofilms were fixed and stained with SYTO 9 green fluorescent stain prior to confocal microscopy analysis. Representative micrographs comparing biofilm thickness of each Tsukamurella strain was measured in different points of each field. The means and standard deviations of three independent experiments are shown.

Article Snippet: Although over 21 “ Tsukamurella species” have been described, using in silico genome-to-genome comparison as the standard of classification, only 16 Tsukamurella species should be included in this genus according to the current state of the taxonomy at the time of writing ( https://lpsn.dsmz.de/genus/tsukamurella ).

Techniques: Functional Assay, Activity Assay, Cell Culture, Mutagenesis, Quantitation Assay, Staining, Confocal Microscopy