BIODIVERSITAS ISSN: 1412-033X Volume 23, Number 2, February 2022 E-ISSN: 2085-4722 Pages: 962-968 DOI: 10.13057/biodiv/d230239 Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever SRI DARMAWATI 1,2, , STALIS NORMA ETHICA 1 , MUHAMMAD EVY PRASTIYANTO 3 , SULAIMAN NGONGU DEPAMEDE 4 , ELEVENTI OKTARINA PUTRI 1 , MUDYAWATI KAMARUDDIN 1 1 Postgraduate of Laboratory for Clinical Science, Faculty of Nursing and Health Sciences, Universitas Muhammadiyah Semarang. Jl. Kedungmundu Raya No. 18, Semarang 50273, Central Java, Indonesia. Tel.: +62-24-76740296,  email: ciciekdarma@unimus.ac.id 2 Muhammadiyah Research Network for Plasma Medicine (M-Plasmed). Jl. Tidar No. 21, Kota Magelang 59214, Central Java, Indonesia 3 Department of Medical Labolatory Technology, Faculty of Nursing and Health Sciences, Universitas Muhammadiyah Semarang. Jl. Kedungmundu Raya No. 18, Semarang 50273, Central Java, Indonesia 4 Faculty of Animal Science and Consortium for Large Ruminant Research, Universitas Mataram. Jl. Majapahit No. 62, Mataram 83125, West Nusa Tenggara, Indonesia Manuscript received: 3 December 2021. Revision accepted: 25 January 2022. Abstract. Darmawati S, Ethica SN, Prastiyanto ME, Depamede SN, Putri EO, Kamaruddin M. 2022. Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever. Biodiversitas 23: 962-968. Blood culture is the gold standard for diagnosing typhoid fever, but it has limitations such as media and laboratory equipment, specimen volume, and examination time. However, the Academy of Pediatrics does not recommend serology due to its low sensitivity. The purpose of this study was to determine the molecular properties of the protein pilli of Salmonella typhi (S. typhi) that the findings can be used to develop a typhoid fever diagnostic reagent. The SDS-PAGE method was used, as well sequence analysis with ProtParam, ProtScale, and PSIPRED. The SDS- PAGE profile reveals one major protein (42 kDa) and fourteen minor proteins. The pili protein subunit 42 kDa had an amino acid (AA) sequence with a length of 390 AA, according to bioinformatics analysis. According to the ProtParam results, the pili protein subunit 42 kDa has good stability with a value of 40 and is a hydrophilic protein with an average GRAVY value of -0.950. PSIPRED results show that among the secondary structural elements, coil strand predominates, followed by -helix and -strand. It is concluded that this protein is immunogenic and that it can be used to develop a more specific and sensitive diagnostic reagent for typhoid fever. Keywords: Pili protein, Salmonella typhi, molecular characterization, typhoid fever INTRODUCTION Typhoid fever is an infectious condition that spreads throughout the body and is caused by the Gram-negative bacterium Salmonella enterica subspecies enterica serovar typhi (S. typhi) (Thieu et al. 2017; Ajibola et al. 2018). Typhoid fever is usually contracted by ingestion of water or food contaminated by fecal or urinary carriers excreting S. Typhi. This is one of the leading causes of mortality in many underdeveloped countries, including Indonesia. Globally, in 2010 typhoid fever was reported in 26.9 million cases (Buckle et al. 2012). World health organization estimates the incidence of typhoid fever at 21 million cases and approximately 161000 deaths (World Health Organization 2018). In Indonesia, in August 2002 and July 2004 typhoid fever is an endemic disease, with 81.7 cases per 100 000 people per year for children aged 24–60 months 148.7 per 100 000 (Wain et al. 2015). Cases of typhoid fever in the city of Semarang show that there is always an infection every month and is a disease that often occurs in large numbers. Based on the recapitulation of typhoid reports at the Semarang City Health Center, in 2015 there were 6,958 cases while in 2016 there were 7,796 cases (Andayani and Arulita 2018). Children (aged 5 to 15 years of age) are the most affected age group with a peak incidence known to occur in individuals (Pitzer et al. 2014). Due to the high incidence of typhoid in developing countries predominantly in Asia including Indonesia, prevention has become a global health priority (Jamka et al. 2019; Sahastrabuddhe and Saluja 2019). Symptoms of typhoid infection include fever which lasts 1 to 4 weeks. Fever is accompanied by headache, chills, abdominal pain, nausea, and dry cough (Paul and Bandyopadhyay 2017). Typhoid fever commonly exhibits non-specific clinical symptoms comparable to malaria, dengue fever, influenza, leptospirosis, and Rickettsia infection, thus a definite diagnosis must be verified by laboratory tests (Azmatullah et al. 2015; Arora et al. 2019). Blood culture is widely recommended as a method for laboratory diagnosis, however its sensitivity ranges from 40 to 80%, not all laboratories have bacterial culture facilities, it is expensive, and it takes 2-3 days to complete (Ajibola et al. 2018). Serological tests such as Widal are also often employed in laboratories because they are quick, easy, and affordable, but their sensitivity and specificity are problematic due to the frequent sharing of epitopes between the antigens of S. typhi, and other Gram-negative rods (Darmawati et al. 2015). It also makes use of a Rapid Diagnostic Test (RDT) for antibody detection. Typhidot, Typhidot M, Typhi Rapid IgM, IgG IgM (Combo), and Tubex TF (anti LPS antibody detection) are 50kDa outer membrane anti-proteins with a wide range of sensitivity