Genetics and Molecular Biology, 44, 1, E20200029 (2021) Copyright © Sociedade Brasileira De Genética

Genetics and Molecular Biology, 44, 1, e20200029 (2021) Copyright © Sociedade Brasileira de Genética. DOI: https://doi.org/10.1590/1678-4685-GMB-2020-0029 Research Article Genetics of Microorganisms Analysis of potential virulence genes and competence to transformation in Haemophilus influenzae biotype aegyptius associated with Brazilian Purpuric Fever Rafaella Fabiana Carneiro Pereira1, Thais Holtz Theizen1, Daisy Machado1, João Paulo de Oliveira Guarnieri2, Gabriel Piccirillo Gomide2, Luciana Maria de Hollanda1and Marcelo Lancellotti 1,2 1Universidade Estadual de Campinas, Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Campinas, SP, Brazil. 2Universidade Estadual de Campinas - UNICAMP, Faculdade de Ciências Farmacêuticas - FCF, Campinas, SP, Brazil. Abstract Brazilian Purpuric Fever (BPF) is a hemorrhagic pediatric illness caused by Haemophilus influenzae biogroup aegyptius (Hae), a bacterium that was formerly associated with self-limited purulent conjunctivitis. BPF is assumed to be eradicated. However, the virulence mechanisms inherent to Hae strains associated with BPF is still a mystery and deficient in studies. Here, we aim to analyze the role of the autotransporter genes related to adherence and colonization las, tabA1, and hadA genes through RT-qPCR expression profiling and knockout mutants. Relative quantification by real-time PCR after infection in human cells and infant rat model suggests that las was initially downregulated probably duo to immune evasion, tabA1, and hadA were overexpressed in general, suggesting an active role of TabA1 and HadA1 adhesins in Hae in vitro and in vivo. Transformation attempts were unsuccessful despite the use of multiple technical approaches and in silico analysis revealed that Hae lacks genes related to competence in Haemophilus, which could be part of the elucidation of the difficulty of genetically manipulating Hae strains. Keywords: Haemophilus influenzae biotype aegyptius, Brazilian Purpuric Fever, virulence, qPCR, competence. Received: February 21, 2020; Accepted: November 18, 2020. Introduction autotransporters (ATs). Homology and sequence analysis Haemophilus influenzae biogroup aegyptius (Hae) was suggest that las emerged from Hi lav (Davis et al., 2001). formerly associated with seasonal epidemics of self-limited Genome comparative analysis identified that Hae purulent conjunctivitis (“pink eye”) until the 1980s, when accessory genome has 21 specific Hae-BPF putative genes an emergent clone of Hae was identified as the etiological (Strouts et al., 2012; Pereira et al., 2019) and a much richer agent of Brazilian Purpuric Fever (BPF) (Brenner et al., repertoire of AT adhesins in comparison to another available 1988; Harrison et al., 2008). BPF is a fulminant pediatric genome of Haemophilus genus. A total of eight new trimeric disease characterized by conjunctivitis, high fever, purpura, autotransporter adhesins (TAAs) was described in Hae (BPF and sepsis. BPF major outbreaks arose from 1984 to 1990 and conjunctivitis isolates) and are present as homologs, in São Paulo, Brazil. Sporadic cases have been registered termed tabA - for the Hae-BPF alleles, or tahA - for the Hae in Australia, USA (Harrison et al., 2008) and more recently non-BPF alleles. TabA1 was characterized as the P145 protein in 2007 in Pará, Brazil (Santana-Porto et al., 2009). BPF is (Strouts et al., 2012), identified in Hae isolates from animals a disease with mandatory reporting in Brazil. The invasive (Rubin and Rizvi, 1991; Rubin, 1995). The tabA1 and tahA1 unique phenotype highlights Hae associated with BPF (Hae- loci are different in the coding sequences, suggesting different BPF) from other Haemophilus influenzae strains (Hi). functions, and tabA1, possess two additional coding sequences, Potential virulence factors such as pilus proteins and HIBPF06250 and IS1016 (absent in non-BPF strains) (Strouts lipopolysaccharide have been studied in vitro with endothelial et al., 2012). The formerly described as the Haemophilus cells (Quinn et al., 1995; Weyant et al., 1994) and in infant capsulation locus–associated insertion sequence IS1016 has rats (Rubin and St Geme, 1993); however, the origin and been related with unusual and invasive virulence of non- virulence mechanisms of Hae-BPF is still unknown. A potential typeable Hi (NTHI) isolates. pathogenic determinant in Hae is the las gene, a member HadA is a TAA unique to BPF associated strains and of the AIDA-I/VirG/PerT family of virulence-associated belongs to the ‘oligomeric coiled-coil adhesin’ (Oca) family found in virulent bacterial isolates. According to Serruto et al. (2009), hadA gene is missing in Hi Rd KW20 and non -BPF Hae F1947, but these strains present the sequences Send correspondence to Marcelo Lancellotti. Universidade Estadual de Campinas, Departamento de Bioquímica e Biologia Tecidual, flanking this gene. Because the GC composition of hadA is Instituto de Biologia, Rua Monteiro Lobato, 255, 13.083-970, significantly different from the rest of the Hi genomes (Serruto Campinas, SP, Brazil. E-mail: [email protected]. et al., 2009) and the F3031 genome (Strouts et al., 2012), it is 2 Pereira et al. believed hadA has been acquired by horizontal transfer (HT) bovine serum (Gibco, USA). Cells suspensions were seeded in (Serruto et al., 2009). 12-well tissue culture plates and incubated until confluence of Since HT is a crucial element in the evolution and cell monolayers. A total of 50 µl of the bacterial suspensions emergence of bacterial pathogens, a possible explanation for with approximately 1.107 CFU of F3031, F3033, KC1018, and the clonal emergence of Hae-BPF is the acquisition of one or ATCC11116 strains were inoculated in). After an incubation more virulence genes by HT (Bartlett, 2006, Diep et al., 2006). time of 6 hours at 37 °C with 5 % CO2, adherent bacteria were Hi is a bacterium naturally competent to transformation (Mell recovered in chocolate agar, grown as described previously et al., 2011, Sinha et al., 2012). Sinha et al. (2012) identified and used for RT-qPCR analysis. All assays were performed the Sxy-dependent cyclic AMP receptor protein (CRP-S) with technical triplicates. regulon required for natural transformation in Hi. The genetic Animal model infection assays manipulation in Hae could be an important tool to elucidate the role of virulence genes in Hae-BPF. However, it was only Litters of Sprague-Dawley rats (Rattus norvegicus) were once registered as successful in the literature (Segada and acquired from “Centro Multidisciplinar para Investigação Lesse, 1997); several attempts were made to obtain mutants Biológica na Área de Ciência em Animais de Laboratório – CEMIB/UNICAMP” (http://www.cemib.unicamp.br). They strains but all failed (Li et al., 2003, Serruto et al., 2009). were bred in a specific-pathogen-free facility at IB-UNICAMP Here, we describe for the first time the expression of and were 8 to 10 days old. The animal model and protocol three potential virulence-associated AT genes of Hae-BPF were based on previous studies with Hae (Rubin and Carlone, in human cells and in the infant rat model. We also tried 1989; Rubin and St Geme, 1993). Briefly, a single dose of to elucidate the puzzling enigma of Hae competence to 1.105 CFU of bacteria was given intraperitoneally and animal transformation. infection was performed in groups as described in Table 2. Material and Methods After a period of 24 and 48 hours, animals were anesthetized by ketamine/xylasin use (50/5 mg/kg) before euthanasia, and Bacterial growth and DNA extraction blood was obtained by heart puncture. Bacteria were recovered in chocolate agar, grown as described previously, and used Growth conditions and DNA extraction protocol for further analysis. followed the methods described previously (Varela et al., The protocol for animal practice was approved 2014; Cury et al., 2014). Bacterial strains used in this work in accordance with relevant guidelines and regulations: are described in Table 1. “Comissão de Ética e Uso de Animais – CEUA – UNICAMP” Comission of Ethics in animal use protocol number: 3197-1 In vitro infection assays (http://www.ib.unicamp.br/comissoes/ceua)”. In vitro infection assays were based on methods described by Pereira et al. (2011). Briefly, A549 (human pulmonary RNA extraction epithelial carcinoma) and Hec1b (human endometrium Bacterial isolates from in vitro and in vivo assays were adenocarcinoma) cells were grown in RPMI-1640 medium grown as previously described. Extraction of total RNA was (Cultilab, Campinas, Brazil) supplemented with 10 % of fetal in Trizol Reagent® protocol. RNA concentration and quality Table 1 – Bacterial strains used in this work. Strain Alias Bacteria GenBank acession no. F3031 254 Hae-BPF FQ670178 F3033 258 Hae-BPF NA F3030 219 Hae-BPF LNKQ00000000 F3039 321 Hae-BPF LNKN00000000 F3042 284 Hae-BPF LNKO00000000 F3283 406 Hae-BPF LNKP00000000 F1949 Karina Hae-BPF LNKS00000000 F3028 329 Hae-BPF CP043771 F3037 167 Hae-BPF CP043772 F3043 55 Hae CP043811 F3052 232 Hae CP043810 F3047 73 Hae FQ670204 KC1018 – Hae LNKR00000000 ATCC11116 – Hae AFBC00000000 RdKW20 – Hi type d L42023 β-lac Hib-βlac Hi type b NA JM109 – Competent E. coli – Virulence genes in Hae – BPF 3 Table 2 – Recovery of animal-passaged strains from infection assays. Strain No. animals with positive blood culture/ total 24 hours 48 hours F3031 2/5 0/5 F3033 2/5 2/5 KC1018 0/5 0/5 ATCC11116 0/3 – were determined by NanoDrop (NanoDrop® 2000 - Thermo et al., 2002). An expression stability coefficient for candidate

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