Role and Mechanism of Cotrimoxazole Resistance to Talaromyces marneffei fungus in vitro
Authors: Chen Jie , Chen Rongfeng , Wei Wudi , Qin Fengxiang , Chen Xiu , He Jinhao , Zhang Hong , Wang Gang , Shi Minjuan , Qin Tongxue , Liao Yinlu , Wu Yuting , Lu Beibei , Tao Xing , Ye Li , Liang Hao , Jiang Junjun 
Published: April 2022

Abstract
Background Talaromyces marneffei (formerly Penicillium marneffei) is an important thermally dimorphic fungus endemic which is characterized by one of the most frequent opportunistic infections in HIV/AIDS patients, mainly prevalent in Southeast Asia, southern China, and northeastern India. Cotrimoxazole(CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi, thereby commonly used to prevent several opportunistic infections among HIV/AIDS patients. In addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis are considered to have the potential to prevent T.marneffei infection in HIV/AIDS patients receiving antiretroviral therapy (ART). However, the role and mechanism of cotrimoxazole resistance to T.marneffei fungus remains unclear.Methods Human THP-1 macrophages were used as cell model in vitro to explore the role and mechanism of cotrimoxazole resistance towards T.marneffei. Cell viability assay and drug sensitivity colony forming units (CFU) experiments were conducted to determine the minimum inhibitory concentration (MIC) of cotrimoxazole inside and outside THP-1 macrophages respectively. Enzyme-linked immunosorbent assay (Elisa) was used to measure the concentration of Dihydropteroic acid synthetase (DHPS), Dihydrofolate synthetase (DHFS) and Dihydrofolate reductase (DHFR) between T.marneffei adding TMP/SMX and without adding TMP/SMX group respectively. Real-time fluorescence quantitative PCR(qPCR) was performed to detect the mRNA expression levels in Dectin-1 mediated signaling pathway and downstream inflammatory cytokines including IL-6, IL-10, IL-23A, CXCL8 and TNF-α released by T.marneffei-infected macrophages between adding TMP/SMX and without adding TMP/SMX group respectively.Results Cotrimoxazole can inhibit the proliferation of T.marneffei within safe concentration inside and outside THP-1 macrophages. Drug susceptibility results showed the minimal inhibit concentration(MIC) of 1:5 TMP/SMX was ranging from 14/70 to 90/450μg/ml. The MIC of SMX was ranging from 100 to 360μg/ml, The MIC of TMP was ranging from 240 to 400μg/ml outside macrophages. The MIC of TMP/SMX was ranging from 36/180 to 68/340μg/ml. The MIC of SMX was ranging from 340 to 360μg/ml. The MIC of TMP was ranging from 320 to 400μg/ml inside macrophages. The synergistic interaction of 1:5 TMP/SMX was more effective in inhibiting T.marneffei than separate SMX and TMP. DHPS, DHFS and DHFR can be inhibited by cotrimoxazole within safe and effective concentration. Dectin-1 expression is increased following T.marneffei infection, leading to the increase of IL-6, IL-10, IL-23A and the decrease of CXCL8 and TNF-α. Conversely, cotrimoxazole decrease the levels of IL-6, IL-10, IL-23A and increase the levels of CXCL8 and TNF-α, thereby enhancing the intracellular killing-T.marneffei capacity of macrophages.Conclusions Our findings indicated that cotrimoxazole directly inhibited T.marneffei growth by blocking DHPS, DHFS and DHFR and indirectly inhibit T.marneffei growth by regulating the Dectin-1 signaling pathway, which may effectively interfere with the defense ability of the host against T.marneffei infection.