Comparative study of the immune response of human macrophages infected with different strains of the Mycobacterium tuberculosis complex and Mycobacterium canettii

Author(s)

Felipe Silva

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2023-03-10

Examining board members

Ana Marcia de Sá Guimarães; Lucas Miranda Marques; Mariângela de Oliveira Silva

• Advisor: Ana Marcia de Sá Guimarães

Abstract

Tuberculosis (TB) is a human and animal disease caused by bactérias belonging to the Mycobacterium tuberculosis complex (MTBC). Despite high genetic similarity, MTBC pathogens show variance in host predilection and virulence. Those adapted to humans include Mycobacterium tuberculosis (M.tb), distributed worldwide, and Mycobacterium africanum L5 and L6, restricted to West Africa. Among animal-adapted species, Mycobacterium bovis and Mycobacterium caprae standout, the main causatives of bovine and zoonotic TB, capable of infecting several host species, including humans. Another tuberculous mycobacteria of importance, which is not part of MTBC, is Mycobacterium canettii, originating from a common ancestor to MTBC and identified as causing TB in humans in East Africa. These are the most relevant pathogens for human TB, whether the disease is zoonotic or not. Regardless of the bacterial ecotype, the main feature of the pathogenesis of tuberculous mycobacteria is their ability to survive inside macrophages by manipulating microbicidal pathways. Consequently, the intracellular persistence of these bacteria leads to apoptosis or necrosis of macrophages, contributing to the control or dissemination of the infection. Several studies evaluated the interaction of M.tb with macrophages and its intracellular persistence, however, few include other MTBC representatives. Thus, the objectives of this study were initially (i) to develop a methodology for quantification of MTBC species (M.tb, M. africanum L5 and L6, M. bovis, M. caprae) and M. canettii by flow cytometry, to facilitate the development of experiments and then (ii) to compare the dynamics of infection of MTBC and M. canettii species in THP-1 macrophages evaluating the infection rate, phagocytosis index and cell death. First, we verified that the quantification by CFU (colony forming units) of M.tb in solid media Middlebrook 7H10 or 7H9-Agar supplemented with 10% OADC (oleic acid, albumin, dextrose and catalase) and 18 mM of sodium pyruvate it is about 10 times larger than the other MTBC species and M. canettii. To understand this discrepancy, the LSR FortessaTM Cell Analyzer flow cytometer (BD Biosciences) was used to quantify bacteria cultured in liquid medium comparing two fluorescence methodologies: staining bacteria with CF-SE (5-(e-6) carboxyfluorescein, succinylmidyl ester ) and transformation with plasmid pMSP::dsRed2. While CF-SE marked only 40 to 80% of bacteria, dsRed2 was expressed in ˜ 100% of bacterial populations. Bacteria- dsRed2 could then be accurately quantified by flow cytometry after adjusting the threshold of PE (phycoerythrin) and PMT voltage (photomultiplier tube) of FSC (forward scatter) to reduce equipment oscillation and the rate of abort events, with low false discovery rate (FDR) and coefficients of variations between replicates. With the new methodology, quantifications by CFU and flow cytometry were equivalent for M.tb, but significantly different in the other evaluated species, indicating that the solid medium is not able to support the adequate growth of non-M.tb species . Then, using the new bacterial quantification protocol, we compared the infection rates and phagocytosis rates in THP-1 macrophages infected with MTBC and M. canettii using different multiplicities of infection (MOIs of 2:1, 5:1, 10:1 and 20:1). Both indices behaved in a dose-dependent manner for all species, except for M. caprae, which presented similar indices in all MOIs, at levels that were only obtained in higher MOIs (10:1 and 20:1) by other MTBC species. Additionally, M. canettii indices significantly outperformed the MTBC species in all MOIs. To explain this finding, we sequenced the complete genome of M. canettii and showed that the tested strain lost one pks5 gene copie (polyketide synthase 5) responsible for the synthesis of lipooligosaccharides. Spontaneous loss of this gene has been associated with greater infectivity of M. canettii in macrophages, explaining the findings. Finally, the levels of cell death evaluated during 96h indicate that M. bovis and M. caprae induce higher levels of apoptosis 24h p.i. in THP-1 macrophages. As the infection progresses, necrosis levels increase, however M. caprae outperformed all species at 72h and 96h of infection. In conclusion, this is a comparative study between MTBC species that brings important methodological contributions and to the understanding of macrophage infection by MTBC species and M. canettii. (AU)

• FAPESP's process: 19/21847-8 - Comparative study of the immune response of human macrophages infected with different strains of the Mycobacterium tuberculosis complex
• Grantee: Felipe Silva
• Support Opportunities: Scholarships in Brazil - Master