Abstract Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata – a major intracellular fungal pathogen – produces small and slow-growing respiratory-deficient colonies, termed “petite.” Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0–3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called “petite”. This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

OverlookedCandida glabratapetites are echinocandin tolerant, induce host inflammatory responses, and display poorin vivofitness