In vivo RNA-seq and infection model reveal the different infection and immune characteristics of B. pertussis strains in China

Li Shi^1*Weilun Zuo^1,2Chen Wei³Meiyan He⁴Mengyao Zhang¹Jiangli Liang⁵Xiao Ma³Na Gao⁵Qin Gu⁵Yan Ma⁵Jingyan Li⁵Shuyuan Liu¹Yan Huang⁴Mingbo Sun^5*

• ¹Laboratory of Immunogenetics, Chinese Academy of Medical Science & Peking Union Medical College, Institute of Medical Biology, Kunming, China
• ²Department of Laboratory Medicine, Fujian Key Clinical Specialty of Laboratory Medicine, Women and Children’s Hospital, School of Medicine, Xiamen University, Xiamen, Fujian Province, China
• ³NHC Key Laboratory of Research on Quality and Standardization of Biotech Products & NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control (China), Beijing, Beijing Municipality, China
• ⁴Department of Pulmonary and Critical Care Medicine, Xishuangbanna Dai Autonomous Prefecture People's Hospital, Jinhong, Yunnan, China
• ⁵Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Chinese Academy of Medical Science & Peking Union Medical College, Institute of Medical Biology, Kunming, Yunnan Province, China

Introduction: Various strains emerged in B. pertussis re-emergence, the pathogenic characteristics and mechanisms remain elusive. We aimed to explore the relationship between the in-vivo transcriptome and colonization advantage of various pertussis clinical strains during the B. pertussis re-emergence.Methods: Four pertussis strains were isolated from clinically suspected cases by active surveillance. The phylogenetic relationships of clinical strains and global isolates were compared by a genome-wide SNP-based phylogenetic tree and allele genotyping. LC-MS/MS analysis and binding affinity detection allowed the identification of expression and antigenicity of pertactin.The characteristics of infection and immunity of clinical strains were compared in a BALB/c mouse aerosol challenge model. In vivo RNA-seq analysis was performed in NSG mouse model to describe the transcriptome during infection, and verified by detecting biofilm formation and paraquat tolerance.The partial pertactin-deficient strain BP-L2 was first reported. It showed significantly enhanced tracheal colonization compared to both CS and BP-L1 strains in naive mice (P <0.0001 vs. CS) and exhibited superior fitness over BP-L1 in immunized mice (P <0.001). BP-L1 showed superior lung colonization (P < 0.0001) and tissue-resident memory T cell induction versus BP-L2 and CS (P <0.001). Colonization dominance of BP-L1 in lungs and BP-L2 in trachea aligned with the pathological injury (P < 0.05) and the inflammatory cytokine enhancement (IL-6 in lungs of BP-L1 group, P <0.01). In vivo RNA-seq results revealed that BP-L2 significantly upregulated relA (log2FC = 2.1, FDR P-value = 0.019) and sodA (log2FC =2.4, FDR P-value = 8.61E-06) compared to BP-L1, functionally linked to enhanced stringent response and oxidative stress defense. BP-L1 exhibited significant in vivo bipA upregulation over BP-L2 (log2FC = 1.8, FDR P-value = 0.027) without concurrent biofilm enhancement (P = 0.51 vs. BP-L2). Furthermore, the BP-L2 and BP-L3 strains of the same ptxP1-ptxA1-fhaB3 lineage showed significantly higher paraquat tolerance than other strains (P <0.001), showing extremely high SODs activity.The emerging pertussis strains exhibit different colonization advantages in the trachea or lungs, which will influence the transmission patterns of the clinical strains. The tracheal colonization advantage of the partial pertactin-deficient strain may be associated with the overexpression of the relA and sodA in vivo infection.