Efeito da deleção do gene mgtC sobre a patogenicidade de Salmonella Gallinarum em aves susceptíveis
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2022-05-31
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Universidade Estadual Paulista (Unesp)
Resumo
Salmonella Gallinarum (SG) causa o tifo aviário, doença infecciosa de curso clínico agudo que acomete galináceos de qualquer idade e que resulta em elevada taxa de mortalidade. Durante a infecção sistêmica do quadro tifoide causado por Salmonella Typhimurium (STM) em camundongos, a bactéria expressa o gene mgtC, presente na Ilha de Patogenicidade de Salmonella – 3 (SPI-3). Sua função está aparentemente ligada à multiplicação bacteriana no interior de macrófagos e sua inativação atenua o patógeno. Dessa forma, hipotetizou-se que sua deleção do genoma de SG alteraria a patogenicidade do microrganismo em aves comerciais susceptíveis visto que a infecção sistêmica cumpre papel crucial para o patógeno. Assim, o presente estudo objetivou elucidar a importância do gene mgtC para a patogenicidade de SG. Para isso, foi construída uma estirpe mutante de Salmonella Gallinarum defectiva para o gene mgtC (SG ∆mgtC). Posteriormente, foi avaliada sua capacidade de multiplicação em meio de cultivo que mimetiza o ambiente intracelular dos macrófagos e também nesses tipos de células imunes. Além disso, foi conduzida a infecção com SG ∆mgtC e pela estirpe selvagem de aves susceptíveis para comparar a patogenicidade, infecção sistêmica e a resposta imune induzidas por meio da mensuração de mRNA das citocinas IFN-γ e LITAF por RT-qPCR e pela quantificação da população de macrófagos e linfócitos T CD4+ e CD8+ por imunohistoquímica. Foi observado que mgtC foi necessário na multiplicação de Salmonella Gallinarum em meio acidificado com baixa concentração de Mg2+ e em macrófagos. Porém, só houve menor contagem bacteriana na fase tardia da infecção, sem afetar a citotoxicidade. Experimentos in vivo demonstraram que a deleção de mgtC não alterou a capacidade de invasão bacteriana e nem a contagem microbiana em fígado e baço e a mortalidade total das aves. Entretanto, ao realizar a curva de sobrevivência e analisar a evolução do quadro clínico-patológico notou-se uma progressão mais lenta da doença em aves infectadas com SG ∆mgtC em comparação com aquelas desafiadas com a estirpe selvagem. Além disso, foi necessário 1,36 Log10 de UFC/mL a mais de bactérias mutantes para causar 50 % de mortalidade em comparação com a estirpe selvagem. Ademais, a expressão de mRNA de IFN-γ e LITAF foi semelhante entre grupos infectados, mas maior em relação aos não-infectados. O mesmo foi observado nas populações de macrófagos e linfócitos T CD4+. Por outro lado, a presença de linfócitos T CD8+ foi maior na fase inical da doença provocado pela estirpe selvagem. O papel de mgtC no tifo aviário difere para infecções tifoides em mamíferos, visto que sua deleção atenuou outros sorovares. Assim, a deleção do gene mgtC do genoma de Salmonella Gallinarum não afeta a patogenicidade, mas altera levemente a patogênese.
Salmonella Gallinarum (SG) provokes the so-called Fowl Typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection caused by Salmonella Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is clustered in the Salmonella Pathogenecity Island – 3 (SPI-3). Its function is apparently linked to bacterial replication within macrophages and its absence attenuates the pathogen. In this sense, it was hypothesized that knocking-out mgtC from SG genome could alter the microorganism pathogenicity in susceptible commercial poultry taking into account the central role of systemic infection to the pathogen. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-lacking Salmonella Gallinarum mutant was constructed (SG ∆mgtC). In addition, its ability to replicate in medium that mimicries the intracellular environment of macrophages and also in this immune cell types was evaluated. Moreover, the infection of susceptible chickens by SG ∆mgtC and the wild-type strain was performed to compare the pathogenicity, systemic infection and the elicited immune responses by measuring mRNA of IFN-γ and LITAF cytokines by RT-qPCR and the population of macrophages and lymphocytes T CD4+ and CD8+ by means of immunohistochemistry. It was observed herein that mgtC was required for Salmonella Gallinarum replication in both acidified low-Mg2+ media and macrophages. Nevertheless, lower bacterial counts was only noticed at the late stage of infection without affecting the citotoxicity. In vivo experiments showed that knocking-out the mgtC gene neither alters bacterial invasiveness ability nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analysing the development of clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ∆mgtC compared to those challenged by the wild-type strain. Furthermore, it was required more 1.36 Log10 of CFU/mL of mutant strain to provoke 50 % of mortality in comparison to the wild-type strain. Moreover, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than those uninfected. The same was observed in macrophages and lymphocytes T CD4+ populations. On the other hand, the presence of lymphocytes T CD8+ was higher in the initial phase of the disease provoked by the wild-type. The role of mgtC in Fowl Typhoid in susceptible chickens differs from the typhoid-like infections in mammals, since its depletion attenuated other serovars. Thus, the deletion of mgtC gene from Salmonella Gallinarum genome does not affect the pathogenicity, but slightly alters the pathogenesis.
Salmonella Gallinarum (SG) provokes the so-called Fowl Typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection caused by Salmonella Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is clustered in the Salmonella Pathogenecity Island – 3 (SPI-3). Its function is apparently linked to bacterial replication within macrophages and its absence attenuates the pathogen. In this sense, it was hypothesized that knocking-out mgtC from SG genome could alter the microorganism pathogenicity in susceptible commercial poultry taking into account the central role of systemic infection to the pathogen. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-lacking Salmonella Gallinarum mutant was constructed (SG ∆mgtC). In addition, its ability to replicate in medium that mimicries the intracellular environment of macrophages and also in this immune cell types was evaluated. Moreover, the infection of susceptible chickens by SG ∆mgtC and the wild-type strain was performed to compare the pathogenicity, systemic infection and the elicited immune responses by measuring mRNA of IFN-γ and LITAF cytokines by RT-qPCR and the population of macrophages and lymphocytes T CD4+ and CD8+ by means of immunohistochemistry. It was observed herein that mgtC was required for Salmonella Gallinarum replication in both acidified low-Mg2+ media and macrophages. Nevertheless, lower bacterial counts was only noticed at the late stage of infection without affecting the citotoxicity. In vivo experiments showed that knocking-out the mgtC gene neither alters bacterial invasiveness ability nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analysing the development of clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ∆mgtC compared to those challenged by the wild-type strain. Furthermore, it was required more 1.36 Log10 of CFU/mL of mutant strain to provoke 50 % of mortality in comparison to the wild-type strain. Moreover, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than those uninfected. The same was observed in macrophages and lymphocytes T CD4+ populations. On the other hand, the presence of lymphocytes T CD8+ was higher in the initial phase of the disease provoked by the wild-type. The role of mgtC in Fowl Typhoid in susceptible chickens differs from the typhoid-like infections in mammals, since its depletion attenuated other serovars. Thus, the deletion of mgtC gene from Salmonella Gallinarum genome does not affect the pathogenicity, but slightly alters the pathogenesis.
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Palavras-chave
Macrófagos, Patogênese, Salmonelose, Fowl typhoid, Macrophages, Pathogenesis, Salmonellosis