The Virulence Factor Ycho Has A Pleiotropic Action In An Avian Pathogenic Escherichia Coli (apec) Strain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Avian pathogenic Escherichia coli strains cause extraintestinal diseases in birds, leading to substantial economic losses to the poultry industry worldwide. Bacteria that invade cells can overcome the host humoral immune response, resulting in a higher pathogenicity potential. Invasins are members of a large family of outer membrane proteins that allow pathogen invasion into host cells by interacting with specific receptors on the cell surface. Results: An in silico analysis of the genome of a septicemic APEC strain (SepT362) demonstrated the presence of a putative invasin homologous to the ychO gene from E. coli str. K-12 substr. MG1655. In vitro and in vivo assays comparing a mutant strain carrying a null mutation of this gene, a complemented strain, and its counterpart wildtype strain showed that ychO plays a role in the pathogenicity of APEC strain SepT362. In vitro assays demonstrated that the mutant strain exhibited significant decreases in bacterial adhesiveness and invasiveness in chicken cells and biofilm formation. In vivo assay indicated a decrease in pathogenicity of the mutant strain. Moreover, transcriptome analysis demonstrated that the ychO deletion affected the expression of 426 genes. Among the altered genes, 93.66 % were downregulated in the mutant, including membrane proteins and metabolism genes. Conclusion: The results led us to propose that gene ychO contributes to the pathogenicity of APEC strain SepT362 influencing, in a pleiotropic manner, many biological characteristics, such as adhesion and invasion of in vitro cultured cells, biofilm formation and motility, which could be due to the possible membrane location of this protein. All of these results suggest that the absence of gene ychO would influence the virulence of the APEC strain herein studied.1635Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [2012/04391-1, 2012/09655-7]CNPqFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration

Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed

Satellite cells from dystrophic muscle retain regenerative capacity

Duchenne muscular dystrophy is an inherited disorder that is characterized by progressive skeletal muscle weakness and wasting, with a failure of muscle maintenance/repair mediated by satellite cells (muscle stem cells). The function of skeletal muscle stem cells resident in dystrophic muscle may be perturbed by being in an increasing pathogenic environment, coupled with constant demands for repairing muscle. To investigate the contribution of satellite cell exhaustion to this process, we tested the functionality of satellite cells isolated from the mdx mouse model of Duchenne muscular dystrophy. We found that satellite cells derived from young mdx mice contributed efficiently to muscle regeneration within our in vivo mouse model. To then test the effects of long-term residence in a dystrophic environment, satellite cells were isolated from aged mdx muscle. Surprisingly, they were as functional as those derived from young or aged wild type donors. Removing satellite cells from a dystrophic milieu reveals that their regenerative capacity remains both intact and similar to satellite cells derived from healthy muscle, indicating that the host environment is critical for controlling satellite cell function

Grafting of a single donor myofibre promotes hypertrophy in dystrophic mouse muscle.

Skeletal muscle has a remarkable capability of regeneration following injury. Satellite cells, the principal muscle stem cells, are responsible for this process. However, this regenerative capacity is reduced in muscular dystrophies or in old age: in both these situations, there is a net loss of muscle fibres. Promoting skeletal muscle muscle hypertrophy could therefore have potential applications for treating muscular dystrophies or sarcopenia. Here, we observed that muscles of dystrophic mdx nude host mice that had been acutely injured by myotoxin and grafted with a single myofibre derived from a normal donor mouse exhibited increased muscle area. Transplantation experiments revealed that the hypertrophic effect is mediated by the grafted fibre and does not require either an imposed injury to the host muscle, or the contribution of donor cells to the host muscle. These results suggest the presence of a crucial cross-talk between the donor fibre and the host muscle environment

Single myofibres grafted into BaCl2-treated host muscles give rise to no donor-derived muscle formation but cause muscle hypertrophy.

<p>Single fibres (SF) isolated from a 3F-nlacZ-2E donor mouse were grafted into TA muscles that had been either irradiated 3 days before (n = 6) (A-I), or that had been BaCl2-injected three days before (n = 10) (A-II); as a control, DMEM was injected into untreated TA muscles (n = 10) (A-III). Donor-derived myofibres (identified by dystrophin expression and incorporation of X-gal positive myonuclei 4 weeks after grafting) were clearly observed in pre-irradiated grafted muscles (I), but to a trivial extent in BaCl2-injured grafted muscles (II) (B), as shown by representative pictures of dystrophin positive fibres with donor-derived myonuclei X-gal stained in serial sections (C and D respectively for I and II). H&E staining of whole transverse-sections from the largest middle part of grafted TA muscles highlighted the difference in size between muscles in I (E) and II (F). This difference was quantified in (G) showing that the cross-sectional area (CSA) of BaCl2-injured and SF-grafted muscles (II) was significantly bigger than in I and III. The number of fibres was not increased in II compared to control, but a loss of fibres was detected in I (H). Size bar = 100 µm. *p<0.05; ***p<0.0001.</p

A donor fibre is required for the hypertrophic effect.

<p>BaCl₂-injured muscles were grafted 3 days later with single fibres (n = 8) (A−I), satellite cells (n = 6) (A−II), or DMEM (n = 6) (A−IV); as a control, irradiated muscles were grafted 3 days later with satellite cells (n = 6) (A−III). As fibres and satellite cells were obtained from β-actin-Cre:R26NZG donor mice (n = 2), their in vivo survival and integration in the recipient host muscles outside myofibres could also be determined. This was quantified alongside the presence of donor-derived dystrophin positive fibres (B). As shown by representative pictures, X-gal positive donor-derived nuclei were found in both BaCl₂-injured (II) and irradiated (III) cell-grafted muscles, inside or nearby the donor-derived dystrophin positive myofibres (C and D respectively). Weights of muscles grafted with fibres (I) were significantly greater than muscles injected with BaCl₂ and DMEM (IV) or irradiated and cell grafted host muscles (III) (E). This increase in size was mirrored by the increased CSA (F), whilst the total number of fibres was not significantly different from the control (IV) (G). Size bar = 100 µm. *p<0.05; **p<0.01; ***p<0.0001.</p

A single donor myofibre injected into recipient mouse muscles promotes muscle hypertrophy.

<p>Single fibres were grafted in mdx nude mouse muscles that had either been injured 3 days previously with BaCl₂ (n = 6) (A-I), or were non-injured (n = 6) (A-II). As a control, DMEM was injected in muscles similarly injured (n = 5) (A-III) or uninjured (n = 5) (A-IV). Representative laminin-stained transverse muscle sections clearly showed that muscles grafted with single fibres (B-I and –II) were macroscopically larger than muscles injected with DMEM (B-III and –IV). This difference was also evident in the weights of the muscles (C). The mean CSA was significantly bigger in muscles in group I compared to III and II compared to IV (D). The number of fibres was not significantly different in any of the cases (E) whilst the distribution of the fibres was changed in muscles injected with single fibres (F) (Chi-squared test: p<0.0001 both when distribution I was compared to III and distribution II was compared to IV). Size bar = 100 µm. *p<0.05.</p

Injection of BaCl2 does not cause muscle hypertrophy.

<p>Mdx nude mice (n = 4) had their right TA injected with BaCl₂ and the left TA with PBS. Laminin-stained transverse sections showed no difference in size between muscles treated in these ways (A). Weights of the muscles were comparable (B) as was the CSA (C). The number of fibres was not significantly different (D) and the distribution of the fibre size was similar (E) (Chi-squared test, p = 0.2261). Size bar = 100 µm.</p